cancers
Review
DNA Damage Responses in Tumors Are Not Proliferative
Stimuli, but Rather They Are DNA Repair Actions Requiring
Supportive Medical Care
Zsuzsanna Suba
Department of Molecular Pathology, National Institute of Oncology, Ráth György Str. 7-9, H-1122 Budapest,
Hungary; subazdr@gmail.com; Tel.: +36-00-36-1-224-86-00; Fax: +36-00-36-1-224-86-20
Simple Summary: This work challenges the traditional principles of cancer therapy: simply targeting
and blocking the regulatory pathways of rapidly proliferating tumors is a serious mistake. Since tumor
initiation and growth may be attributed to a patient’s genomic instability and damage, genotoxic
medications are inappropriate as they cause additional genomic damage in both patients and their
cancers. Tumor cells are not enemies to be killed, but rather they are ill human cells which have the
remnants of same genome stabilizer pathways like healthy cells. Within tumors, there is a combat for
the improvement of their genomic defects. Moreover, tumors ask for help in their kamikaze action by
recruiting immune competent cells into their environment. We should learn by watching the genome
repairing activities within tumors, in the peritumoral region and in the whole body, and may follow
them with supportive care. Successful cancer therapy does not remain a dream to be realized in the
far future, but we should set about a cancer cure without delay.
Citation: Suba, Z. DNA Damage
Responses in Tumors Are Not
Proliferative Stimuli, but Rather They
Are DNA Repair Actions Requiring
Supportive Medical Care. Cancers
2024, 16, 1573. https://doi.org/
10.3390/cancers16081573
Academic Editors: Hassan Bousbaa
and Zhiwei Hu
Received: 5 March 2024
Revised: 5 April 2024
Accepted: 16 April 2024
Published: 19 April 2024
Copyright: © 2024 by the author.
Licensee MDPI, Basel, Switzerland.
Abstract: Background: In tumors, somatic mutagenesis presumably drives the DNA damage response
(DDR) via altered regulatory pathways, increasing genomic instability and proliferative activity. These
considerations led to the standard therapeutic strategy against cancer: the disruption of mutationactivated DNA repair pathways of tumors.Purpose: Justifying that cancer cells are not enemies to be
killed, but rather that they are ill human cells which have the remnants of physiologic regulatory
pathways. Results: 1. Genomic instability and cancer development may be originated from a flaw in
estrogen signaling rather than excessive estrogen signaling; 2. Healthy cells with genomic instability
exhibit somatic mutations, helping DNA restitution; 3. Somatic mutations in tumor cells aim for
the restoration of DNA damage, rather than further genomic derangement; 4. In tumors, estrogen
signaling drives the pathways of DNA stabilization, leading to apoptotic death; 5. In peritumoral
cellular infiltration, the genomic damage of the tumor induces inflammatory cytokine secretion
and increased estrogen synthesis. In the inflammatory cells, an increased growth factor receptor
(GFR) signaling confers the unliganded activation of estrogen receptors (ERs); 6. In breast cancer
cells responsive to genotoxic therapy, constitutive mutations help the upregulation of estrogen
signaling and consequential apoptosis. In breast tumors non-responsive to genotoxic therapy, the
possibilities for ER activation via either liganded or unliganded pathways are exhausted, leading
to farther genomic instability and unrestrained proliferation. Conclusions: Understanding the real
character and behavior of human tumors at the molecular level suggests that we should learn the
genome repairing methods of tumors and follow them by supportive therapy, rather than provoking
additional genomic damages.
Keywords: anti-estrogen; cancer therapy; estrogen; DNA damage; DNA damage response; DNA
repair; endocrine disruptor; estrogen receptor; growth factor receptor; mutation
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Cancer is a complex disease, presumably originating from mutations in genes, promoting genomic instability, and initiating cancer development [1]. In cancers, mutagenesis
Cancers 2024, 16, 1573. https://doi.org/10.3390/cancers16081573
https://www.mdpi.com/journal/cancers
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drives the DNA damage response (DDR) via altered regulatory pathways, increasing genomic instability and helping proliferative activity [2]. Altered DNA damage responses in
tumors serve the maintenance of survival and unrestrained proliferative activity of cells.
These considerations led to the standard therapeutic strategy against cancer: the disruption
of mutation-activated DNA repair pathways of tumors, which should lead to the clinical
recovery of cancer patients [3]. However, the derangement of the mutation-driven DNA
repair techniques of tumors could not bridge the gap between basic research and clinical
practice.
In tumors, the accumulation of somatic mutations yields so-called cancer driver
genes, and their altered regulatory protein products may manage aggressive expansion [4].
Catalogues of genes known to be involved in cancer development were prepared by wholeexome and later, whole-genome sequencing of numerous tumor samples. Analyses of
thousands of cancer genomes return a remarkably similar catalogue of around 300 genes
that are mutated in at least one cancer type. Yet, many features of these mutated genes
and their exact role in cancer development remain unclear. The accumulation of certain
mutated genes in tumors is not enough to justify their pro-oncogenic nature.
There is a close collaboration between the activity of the immune system and cancer
driver mutations. The immune system has a strong impact on determining the expression
of certain cancer driver genes [5]. At the same time, the appearance of certain cancer driver
mutations shows correlations with the density and composition of immune competent
cells in the tumor microenvironment [6]. The connection of the immune system with the
appearance of cancer driver mutations is probably mediated by the fact that all somatic
mutations can create neoantigens. These unknown peptides may trigger an immune
response, eliminating the cell that carries them; this process is known as immune-editing [5].
Cancer driver mutations influence the quantity and composition of immune cell
infiltration in the tumor microenvironment [6]. Somatic mutations in cancer driver genes
with well-known roles in immune signaling, such as CASP8 or HLA, generally recruit higher
concentrations of immune cells into the tumor microenvironment. These pro-oncogenic
mutations most likely result in immune-evading mechanisms. In contrast, colorectal tumors,
with accumulated KRAS mutation, show weaker immune cell infiltration than those without
this mutation, and the tumors are resistant to the immune-checkpoint blockade [7].
Surprisingly, cancer driver genes are exposed, even in various healthy cells exhibiting
the same somatic mutations as tumors. Two studies examined somatic mutations in the
entire human body [8,9]. In some individuals, cancer driver somatic mutations were found
in virtually all tissues, although none of them had been diagnosed with cancer. The most
interesting recent finding is the presence of somatic PTEN, KMT2D, and ARID1A mutations
in healthy liver cells [10]. Hepatocytes showing these well-known cancer driver mutations
exhibited conspicuously increased fitness, faster expansion, and regeneration under stress
or other injury as compared to their counterparts without mutation.
The study on liver cells showing high fitness and regenerative capacity despite their
cancer driving mutation justifies the positive impact of somatic mutations on genomic
stability rather than tumor promotion. There is a plausible explanation; the concentration
of genome driver somatic mutations in tumors may not be pro-oncogenic stimuli, but may
rather be DNA stabilizer actions via genomic plasticity. Somatic mutations in clinically
cancer-free patients may derive from the earlier occurrence of accidental genomic instability
or subclinical cancer in an organ, which were repaired or eliminated via activated mutations.
Molecular cancer therapies targeting the altered DNA damage response pathways lead
to continuous failures. This problem evokes the idea that some modern cancer therapies
might cause more harm than benefit, as we do not exactly understand the molecular events
in the background of diseases [11]. The analysis of therapeutic failures urges a complete
turn in our anti-cancer strategy rather than farther developing and improving the families
of moderately effective or even genotoxic drugs.
The aim of this study is to justify that tumor cells are not enemies to be killed, butrather
that they are ill human cells which have the remnants of the same regulatory pathways
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like patients’ healthy cells [12]. Understanding the real character and behavior of human
tumors at the molecular level suggests that we should learn by watching the genome
repairing methods of tumors instead of provoking additional genomic damages.
2. Endocrine Disruptor Synthetic Estrogens Increase the Risk for Certain Cancers and
Cardiovascular Complications
In the early 1940s, synthetic estrogens were developed for medical purposes; for
the treatment of miscarriage and menopausal complaints and later, for oral contraception. Diethylstilbestrol (DES) was a non-steroidal hormone; ethinylestradiol (EE) was a
steroidal product; while conjugated equine estrogens (CEEs) were extracted from biological
samples [13].
Increased breast cancer risk in DES-treated patients mistakenly suggested that synthetic estrogens activate the same subcellular pathways that a high endogenous estradiol level does, leading to alterations in all cellular functions including interactions with
DNA [14]. In reality, malformations and increased breast cancer risk induced by prenatal
exposure to DES may be attributed to the deregulation of estrogen signaling pathways. In
animal experiments, DES and EE treatment provoked histone modification and further
genomic damages via ER deregulation, justifying their endocrine disruptor character [15].
The development of synthetic estrogens, including both DES and EE, may be regarded
as a pharmaceutical mistake as they are endocrine disruptors. Endocrine disruptors exhibit a special toxicological mechanism; higher doses induce more genomic damages as
compared to lower doses; however, there are no safety low levels of these chemicals [16].
Low doses of synthetic estrogens exert an inhibitory effect on the ligand independent,
ancient AF1 domain of ERs, while inducing compensatory estrogen-like activation on the
ligand-dependent AF2 domain. Conversely, high doses of synthetic estrogens provoke a
serious imbalance between the liganded and unliganded activation of ERs, resulting in
uncompensated damages in the whole genomic machinery [17].
2.1. Controversial Correlations between Menopausal Hormone Therapy (MHT) and
Women’s Health
For menopausal hormone therapy (MHT), both synthetic EE and CEE extracted from
biological samples were prescribed [13]. From the 1940s, MHT became widely used
among postmenopausal women for the treatment of menopausal symptoms and for the
prevention of chronic illnesses, such as cardiovascular and thromboembolic complications
and osteoporosis. In menopausal women, both natural and synthetic estrogens were
applied alone or in combination with synthetic progestins as exogenous hormone therapies.
Among HRT-using women, ambiguous clinical results were experienced; either increased
or decreased risks for arterial and venous thromboembolism and for breast cancer was
experienced. The guidance from the Food and Drug Administration (FDA) established that
the benefits of MHT use surpass their risks [18]. Nevertheless, no comparative information
was available on the efficacy and toxicity of synthetic versus natural hormone products.
In the early 2000s, two great Women’s Health Initiative (WHI) studies reported quite
controversial results in women who underwent MHT. In 2002, increased risks for breast
cancer, thromboembolism, and cardiovascular diseases were reported in menopausal
women treated with conjugated equine estrogen (CEE) plus medroxyprogesterone acetate
(MPA) [19]. Conversely, in 2004, another great WHI study reported on a striking reduction
of breast cancer risk in women treated with CEE (Premarin, Pfizer) alone [20]. In the latter
study, the protective effect of Premarin, with its natural origin, may be explained by the
omission of the highly toxic progestin, MPA [21].
In 2019, a great meta-analysis study reported worldwide epidemiological evidence of
the breast cancer-inducing capacity of MHT independent of the used hormone formuli and
the timing of treatment [22]. All MHT studies reporting the breast cancer preventive effect of
Premarin alone were omitted from this analysis. The concept of “estrogen-induced cancer”
was both the starting point and the goal of investigation, creating a circular reasoning.
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In 2020, the earlier WHI study was repeated on the surviving women eighteen years
following the MHT, and the results reflected the long-lasting breast cancer preventive
effect of Premarin. Both morbidity and breast cancer-associated mortality were significantly
decreased among estrogen treated women [23]. These results justified the long term genome
stabilizer power of natural estrogen treatment without synthetic progestin use [17].
In 2021, Premarin treatment of women with ER positive, PR negative breast cancers
(N = 10,739) resulted in a significant reduction in tumors and breast cancer-related deaths.
The authors established that here is the time for change in their breast cancer risk reduction
strategies in clinical practice [24].
An analysis of the results of MHT studies using different hormone schedules justified
that horse urine-derived Premarin without synthetic progestin is a highly beneficial formula against breast cancer, coronary heart disease, thromboembolism, and bone loss [21].
Although only synthetic hormones may be blamed for increased breast cancer risk and
further complications in MHT-using women, the “estrogen-induced cancer” remained
evidence-based fact.
2.2. Oral Contraceptives Are Endocrine Disruptors Inducing either Increased or Decreased Cancer
Risk in Different Organs
Oral contraceptives (OCs) comprising synthetic EE were developed in the 1960s.
OCs may induce serious toxic side effects, such as venous thromboembolism, stroke, and
cardiovascular diseases [13]. OC use induced the deregulation of ER signaling and led to
an increased risk for insulin resistance and metabolic diseases [25].
Wide spread use of OC use among premenopausal women caused highly ambiguous
correlations with cancer risk at different sites. Among OC user women, a slightly increased
risk for overall breast cancer was observed [26], while strongly increased risks for ER/PR
negative and triple-negative breast cancer (TNBC) were registered [27,28]. Conversely, OC
use significantly reduced the risk of endometrial [29], ovarian [30], and colon cancer [31].
The controversial correlations between OC use and reduced or enhanced cancer risk at
different sites strongly justified that ethinylestradiol is an endocrine disruptor compound
rather than a bioidentical estrogen [17].
In BRCA gene mutation carriers, long term OC use significantly increases the risk
for overall breast cancer as compared to non-carriers [32]. Long term OC use in BRCA
mutation carriers may exert an additional inhibition on the non-liganded ER activation
aggravating mutation associated weakness of ERs. Conversely, in women, with BRCA1/2
gene mutations, the risk for ovarian cancer is strongly reduced by OC use [33] via exerting
an advantageous estrogen-like effect by the indirect activation of the AF2 domain [17].
Despite the known metabolic, thrombotic, and carcinogenic complications of OCs,
they are widely used in medical practice. Clinicians do not believe, or do not want to
believe, in the endocrine-disrupting nature of OCs. In addition, OC use strengthened the
misbelief that endogenous estrogens in higher concentrations may induce increased breast
cancer risk.
3. In BRCA Gene Mutation Carriers, the Defect of Liganded ER Activation Is the
Initiator of DNA Damage and Cancer Development
Patients with the germline BRCA gene mutation are pathological models for genomic
instability and have an increased predisposition for breast and ovarian cancer development.
The first breast cancer gene (BRCA1) was identified in 1994, showing close correlation with
breast cancer development when becoming mutated [34], while the second breast cancer
gene (BRCA2) was announced in 1995 [35]. BRCA1 and BRCA2 genes may be regarded
as safeguards of the genome. Their BRCA protein products control DNA replication,
transcriptional processes, DNA recombination, and the repair of DNA damages [36].
Although functional BRCA proteins have crucial role in the health of all cell types in
both men and women, germline BRCA gene mutations are preferentially associated with
tumor development in female breasts and ovaries [37,38].
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The tissue specificity of BRCA1 mutation-associated tumors suggested a potential
relationship between BRCA1-loss and excessive estrogen signaling in breast cancer development. However, BRCA1 mutation-linked tumors are typically ER-alpha negative,
poorly differentiated, and show rapid growth and poor prognosis [39]. Receptor expression profiling of BRCA1 mutant tumors showed that their vast majority proved to be
ER-alpha negative and ER/PR/HER2 negative, nominated as triple negative breast cancer
(TNBC) [40]. In addition, the development of ER-alpha negative breast cancer has been
reported to be a predictor of BRCA1 mutation status in patients [41]. In sporadic ER-alpha
negative breast cancers, reduced BRCA1 protein expression and a decreased level of ERalpha mRNA were parallel observed, while estrogen treatment increased BRCA1/2 mRNA
levels [42]. These results suggest that BRCA gene mutation deteriorates the regulatory interplay with ERs, leading to decreased ER expression and consequential decreased estrogen
signaling [43].
Since the regulation of healthy female breast requires a strict balance between liganded
and unliganded ER activation, the weakness in ER expression and estrogen activation
results in a preferential susceptibility to genomic damage in the breasts of BRCA mutation
carrier women [43]. In diabetes and obesity, weak estrogen signaling-associated defects
in the hormonal and metabolic equilibrium are directly associated with an increased
TNBC risk.
Molecular studies on the interactions between BRCA1 protein and ER alpha yielded
highly controversial results supporting either the upregulating or downregulating effect of
BRCA1 on ER alpha transactivation.
Wild type BRCA1 gene was demonstrated to inhibit ER alpha transcriptional activity
under the control of its estrogen responsive elements [44]. BRCA1 could suppress the
expression of near all estrogen-regulated genes [45]. In addition, BRCA1 was able to inhibit
p300 mediated ER acetylation, which is essential for the transactivation of ERs [46]. In
contrast, it was reported that BRCA1 may induce an increased transcriptional activity of
ER alpha by the upregulation of p300 expression, a co-activator of ER alpha [47]. Similarly, BRCA1 ensured co-activator Cyclin D binding to ER alpha so as to facilitate the
transcriptional activity [48].
These controversial findings reflect the complexity of regulatory processes, including
both the activation and repression of ERs. In conclusion, estrogen-liganded ER alpha may
choose momentarily appropriate cofactors, promoter regions, and transcriptional pathways
in harmony with optimal BRCA1 expression and activation [49].
In genome stabilization, BRCA and ER proteins are in mutual interaction by direct
binding regulating each other’s activation [50]. The amino-terminus of BRCA1 increases
the activation of ER alpha, while the carboxyl-terminus of BRCA1 may function as a transcriptional repressor on the ER alpha protein. ER alpha and BRCA1 are crucial components
of the regulatory circuit of DNA stabilization as well [49]. Defective expression or activation
of either BRCA1 or ER alpha protein disturbs their interaction, endangering both estrogen
signaling and genomic stability.
In women with the BRCA gene mutation, anovulatory infertility frequently occurs [51],
reflecting the defects of the liganded estrogen signal. In addition, early menopause associated with ovarian failure is also a characteristic finding in BRCA mutation carriers [52]. In
85% of BRCA1 mutation carriers, loss of functional BRCA1 protein correlated with elevated
aromatase levels and increased estrogen synthesis [53] suggesting compensatory actions
against decreased ER expression.
In BRCA mutation carrier breast cells, decreased BRCA1 protein synthesis is associated
with the down-regulation of ER alpha mRNA expression and low ER alpha expression [54].
In BRCA gene mutation carrier tumor cells, a consequently decreased liganded activation
of ERs was observed [44]. In BRCA gene mutation carrier breast cancer cells, a decreased
expression of ER alpha was experienced [55].
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The defect of liganded ER activation in BRCA mutation carriers is a crucial finding, as
it explains the increased inclination for cancers, the ER negativity of developing tumors,
and the ovulatory disorders of female patients.
Both Healthy Cells and Tumor Cells with BRCA Mutation Show Compensatory Molecular
Changes, Improving Genomic Stability
In BRCA mutation carriers, the defect in estrogen signaling endangers the genome
stability in healthy cells, and means a risk for further genomic deregulation in tumor cells.
In healthy cells with BRCA mutation, a compensatory upregulation of estrogen signaling
may preserve genomic stability, while in BRCA mutation carrier tumor cells, increased
estrogen signaling may protect from further genomic damage and increasing proliferative
activity. Tumor cells possess the remnants of the same genome stabilizer pathways like
healthy cells have. In the emergency situation of a weakening estrogen signal, tumor cells
may show various activating mutations, increasing both liganded and unliganded ER
activation [56].
Healthy cells: In mammary epithelial cells, the loss of the BRCA1 gene leads to
increased epidermal growth factor receptor expression [57], which means an unliganded
activation of ERs instead of a pro-oncogenic impact. In BRCA1 mutation carrier women,
BRCA1 protein activity confers the selection of an appropriate CYP19 aromatase promoter
region for the compensatory intensifying of estrogen synthesis [58]. In mammary fibrous
adipose cells, the downregulation of the BRCA1 gene increased the specific activation of
the PII promoter on Cyp19 aromatase gene, leading to increased estrogen synthesis. The
mutation of the BRCA1 gene may be counteracted by the unliganded activation of ERs
via the upregulation of growth factor receptors and P13K/Akt pathways interacting with
BRCA1 protein [59].
Tumor cells: In BRCA1-deficient human ovarian cancer cells, ER alpha exhibited
increased ligand independent transcriptional activity that was not observed in BRCA1
proficient cells [60]. Authors suggested that the loss of BRCA1 increased unliganded ER
activation increasing cancer risk; however, it was a compensatory activation attributed to
the defective liganded activation.
In the tumor cell line with BRCA mutation, increased estrogen signaling was observed
via enhanced activation of p300, a transcriptional coactivator of ERs [47]. In familiar breast
cancers with BRCA mutation, a further transcriptional activator of ERs—Cyclin D1—was
highly accumulated [61]. Nuclear factor kappaB (NF-κB), an important ER coactivator,
was persistently activated in a subset of BRCA1-deficient mammary luminal progenitor
cells [62].
In BRCA1/2 gene mutation carriers, the most frequently co-mutated gene was TP53
(38.1%). Patients with both BRCA1/2 and TP53 gene mutations were more likely to have
hormone receptor negative cancers, high Ki-67 values, and increased genetic mutations,
especially of hormone receptor-related genes. Survival benefits were observed in the
BRCA2 mutation carrier patients with TP53 co-mutation, compared to those with TP53
wild types [63]. This valuable observation supports the increased genome stabilizer impact
of mutated TP53, providing compensatory genome stabilization in tumors with BRCA2
gene mutation.
In sporadic breast cancer cells, the wild BRCA gene is capable of increasing the
expression of the coding gene of ER alpha—ESR1—mediated by the activator Oct-1 [55].
Moreover, BRCA could transcriptionally increase the expression of ER alpha mRNA.
Studies on BRCA mutation carriers teach us crucial new aspects for cancer research:
1. Genomic instability is linked to the weakness of liganded ER activation rather than
excessive estrogen signaling; 2. BRCA gene mutation carrier healthy cells are working
on the improvement of endangered DNA, via the upregulation of both liganded and
unliganded ER activation; 3. In BRCA mutant tumor cells, the upregulation of estrogen
synthesis and unliganded ER activation are efforts to protect DNA from further damage;
4. Both healthy and tumor cells with BRCA gene mutation exhibit gene amplification and
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activate gene mutations so as to increase estrogen synthesis and improve ER activation;
5. In BRCA mutation carriers, the whole body works on genome stabilization via increased
ovarian and peripheral estrogen synthesis.
4. Estrogens Are the Principal Regulators of Genomic Machinery in Mammalian Cells
At the cellular level, estrogen-activated ERs (ER alpha and ER beta) are the hubs of
genomic machinery, orchestrating all cellular functions affecting both somatic and reproductive health [64]. Molecular factors of all cellular processes are working in regulatory
circuits. They receive the regulatory commands from estrogen-activated ERs directly or
indirectly and, at the same time, send their signals back to the ERs closing the circuit.
ER-alpha-regulated DNA stabilizer circuit. ER-alphas activated by the estrogen hormone are the initiators and drivers of the regulatory circuit of DNA stabilization. ERs,
genome safeguarding proteins, such as BRCA1, and estrogen synthesizing aromatase enzyme (A450) create a triangular partnership. The appropriate expression of ER-alpha,
BRCA1 protein, and aromatase enzyme is harmonized by firm interplay among ESR1,
BRCA1, and CYP19 genes and their transcriptional activity in the promoter regions [49]. The
upregulation of estrogen signaling ensures DNA stability in all phases of cell proliferation.
Liganded ER-alpha as a transcriptional factor induces ESR1 gene expression, driving
protein coding ER-alpha-mRNA and ER-alpha protein expression. Liganded ER-alphas are
capable of occupying the BRCA1 gene promoter region as well, facilitating the expression
of BRCA1 mRNA transcripts and increased BRCA1 protein synthesis [37].
The BRCA1 protein, as a transcriptional factor, drives the expression of the BRCA1
gene and amplifies BRCA1 protein expression. The BRCA1 protein activates ESR1 gene
expression and increases ER-alpha protein synthesis [55]. Moreover, the BRCA1 protein is
capable of occupying the promoter region of the CYP19A gene, conferring the augmented
expression of the aromatase enzyme. The BRCA1 protein ensures safety equilibrium between the ER-alpha protein and aromatase enzyme expression [56]. Abundant BRCA1
proteins may induce epigenetic modification and activate mutations on ESR1, BRCA1, and
CYP19 aromatase genes via increasing the appropriate lncRNA expression and resulting in
increased production of the three regulatory proteins: ER, BRCA1, and aromatase [56]. In
addition, abundant BRCA1 proteins are capable of increasing the transcriptional activity
of ER-alpha mediated by either Cyclin D1 [48] or p300 coactivator protein [47]. Increased
BRCA1 activity confers a decreased unliganded activation of ERs [60], while increasing
liganded ER activation and strengthening DNA stability [17]. Some lncRNA transcripts of
BRCA1 may induce transcription on the CYP19 aromatase promoter, facilitating A450 aromatase enzyme expression and estrogen concentration [58]. A high estrogen concentration
helps in the binding and activation of abundant ER-alphas, further stimulating the DNA
stabilizer circuit [49].
The process of estrogen-induced genome stabilization through the ER-BRCA-aromatase
circuit may take many hours as protein synthesis is a time consuming procedure. In emergency situations, 17beta-estradiol can rapidly enhance aromatase enzyme activity and
estrogen synthesis in both healthy and tumor cells. The non-receptor tyrosine kinase
c-Src shows direct involvement in E2 stimulated quick aromatase activation via a short
nongenomic autocrine loop [65].
ER-alpha and BRCA1 proteins can directly bind with each other as transcriptional
factors. Certain binding sites facilitate upregulative processes, while others may quench
each other’s transcriptional activity [50]. Mutagenic defects or the decreased expression of
ER-alpha may dangerously repress the expression of BRCA1 mRNA transcripts and BRCA1protein synthesis; endangering DNA-safeguarding [42]. Similarly, decreased synthesis or
mutagenic alteration of BRCA1-protein results in the downregulation of the expression of
the ER-alpha mRNA and ER-alpha protein [54]. If either the ER-alpha or BRCA1 protein
function suffers damage, the result will be genomic instability and increased cancer risk [49].
ER-alpha-regulated circuit of cell proliferation. The principal regulator of cell proliferation is the ligand-activated ER-alpha in strong interactions with membrane-bound
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tyrosine kinase growth factor receptors; insulin-like growth factor receptor 1 (IGF-1R) and
epidermal growth factor receptor (EGFR) [12]. The equilibrium between liganded and
unliganded ER-alpha activation provides an accurate control over DNA replication in both
high and low phases of cell proliferation. The interplay between ER and GFR receptor
families is the prerequisite of the regulation of cell growth and proliferation and it may be
more or less preserved even in malignant tumors [17].
IGF-1R shows a bidirectional signaling pathway with ligand-activated ERs [66]. IGF-I
expression is influenced by both insulin and growth hormone (GH) stimulating the IGF-I
synthesis in the liver [67]. IGF-1 binding to its receptor, IGF-1R may upregulate two chief
signaling pathways: the phosphatidyloinositol 3-kinase (PI3K-AKT) and the Ras-mitogenactivated protein kinase (MAPK) pathways. These kinase cascades drive the unliganded
transcriptional activity of ER-alpha by the phosphorylation of serine residues [68].
ERs are driving many protein components in the insulin-IGF-1 system, such as the
IGF-1R and insulin receptor substrate 1 (IRS-1) [69]. ER-alpha is capable of binding and
phosphorylating IGF-1R and taking care of its signaling pathways. In IGF-1 KO mice,
estradiol-activated uterine growth is missing [70]. Conversely, in vivo IGF-1 activation of
uterine cell proliferation is strongly dependent on ER-alpha activation [71].
Estrogen stimulates the EGF synthesis in uterine epithelial cells through ER activation,
resulting in a proliferative effect [72]. In estrogen-free milieu, EGFR signaling may be
activated through unliganded ER activation [73]. In turn, in the uterus of ER-alpha KO mice,
EGF could not induce DNA synthesis and transcriptional activity [74]. In ovariectomized
mice, estradiol treatment resulted in a rapid increase in uterine EGFR mRNA and protein
expression and increased the binding sites on EGF through ER activation [75].
In the nucleus, the EGFR signal induces phosphorylation and activation on ER-alpha at
serine 118 location conferred by the growth factor receptor-activated MAPK pathway [76,77].
Phosphorylation at serine 118 increases the ER-associated transactivation of several genes
that are activated by EGFR. The growth factor receptor signal is capable of increasing the
transcriptional activity of nuclear ERs through the phosphorylation of their coactivator
proteins, such as steroid receptor coactivator 1, p300 protein, and cyclin D1 [78,79].
In the cytoplasm, estrogen-activated ERs induce EGFR activation and EGFR conferred
upregulation of the PI3K signaling pathway [80]. In endothelial cells, estrogen treatment
induced PI3K activation resulted in the rapid upregulation of 250 estrogen-regulated genes
within 40 min [81]. The ER/EGFR interplay at the membrane promotes the activation
of numerous signaling pathways that further increases the wide-ranging transcriptional
activity of ERs [66].
In human breast cancer, an inverse correlation may be observed between ER and
EGFR expression [82,83]. In breast cancer cell lines responsive to tamoxifen, a counteractive
increased expression of ERs may be experienced, improving estrogen signaling. In tumors
non-responsive to tamoxifen, an additional increased expression of growth factor receptors
may be experienced [84], conferring the unliganded activation of ERs. Abundant GFRs
highly increase ER activation via unliganded pathway; however, they cannot compensate
the tamoxifen blockade of AF2 domain [17].
ER-alpha-regulated fuel supply circuit. Liganded ER-alpha drives a regulatory circuit
to maintain glucose homeostasis and to stimulate all the phases of cellular glucose uptake
providing fuel for all cellular functions [49]. Defects in the estrogen signal results in serious
alterations in cellular glucose uptake designated as insulin resistance and leads to serious
chronic diseases including cancer [85]. In conclusion, insulin resistance is the linkage
between a weak estrogen signal and increased cancer risk.
Estrogen-regulated genes activate insulin synthesis and secretion, as well as the expression and activation of insulin receptor [86]. When insulin binds to its receptor, autophosphorylations of multiple tyrosines induce the activation of insulin signal transduction [87].
Liganded ERs upregulate the expression and functional activity of intracellular glucose
transporter-4 (GLUT4), promoting insulin-assisted glucose uptake [88]. Liganded ER-alpha
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drives the insulin receptor substrate 1 (IRS1) conferred activation of PI3K/mTOR signaling
pathway which ensures the hormone free activation of nuclear ERs [89].
Estrogen signal activates glucose uptake even in cancer cells supplying energy for the
self-directed improvement of DNA stability. In the MCF-7 breast cancer cell line, estradiol
enhances the expression of the insulin receptor substrate-1 (IRS-1), activating insulin
signaling [90]. In ZR-75-1 breast cancer cells, estrogen/progesterone treatment increased
glucose transporter 1 (GLUT1) expression [91]. In MCF-7 cell lines, estradiol treatment
activated ERs via the PI3K/Akt signaling pathway and, at the same time, increased the
translocation of glucose transporter 4 (GLUT4) vesicles to the plasma membrane [92]. A
defective or blocked estrogen signal results in the failure of glucose uptake even in cancer
cells, declining the activity of genome stabilizer pathways.
5. Estrogens Are Master Regulators of Metabolism and Energy Homeostasis via
Orchestrating Adipose Tissue Functions
Adipose tissue, deposited all over the body, provides energy and epigenetic regulatory
commands for all tissues and organs via its estrogen-activated ER network. In healthy
adipose tissue, estrogen signaling regulates the glucose homeostasis and the balance of
lipolysis/lipogenesis [93,94]. In adipose tissue, damaged estrogen signaling leads to defects
in all regulatory functions, and serious diseases may develop in the fat-regulated visceral
organs, cardiovascular structures, and hemopoietic bone marrow [95].
The subcutaneously located adipose tissue provides energy and estrogen regulation
for the skin and the skeletal muscles. Centrally positioned fatty tissue within the trunk
and abdomen closely surrounds the visceral organs and cardiovascular structures [96].
Visceral fat is largely located in the omental and mesenteric adipose tissue in the vicinity
of stomach, intestines, liver and pancreas. Kidneys, and the attached adrenal glands, are
embedded into abundant fatty tissue capsule. Adipose tissue deposition within the visceral
pericardium surrounds the myocardium and coronary arteries providing estrogen signaling
and energy for the moving heart. Perivascular adipose tissue nurses most blood vessels,
with the exception of the pulmonary and cerebral arteries [97]. A further depot of adipose
tissue is gonadal fat (GAT) surrounding the ovaries and testes having specific regulatory
functions [98].
Female breasts enjoy an exceptional nursing level as mammary lobules are intimately
intermingled with the estrogen and ER rich fatty tissue pad [99]. This close connection
between the adipocytes and mammary cells is associated with the extreme demand of
breasts for strict regulatory control and abundant energy supply. The high claim of breasts
for regulatory commands may explain their unique vulnerability to estrogen loss or defects
in ER activation.
The third largest fat depot is the bone marrow fat, following subcutaneous and visceral
fatty tissue. Adipocytes are active components of the bone marrow microenvironment,
regulating hemopoietic and immune cell proliferation and function via their estrogen signal
and secretome [100].
Interestingly, the central nervous system does not enjoy the estrogen driven adipose
tissue safeguard, while the brain shows an extreme claim for estrogen regulation. Recently,
microbial sequences were found in healthy human brain samples [101] suggesting that they
may provide important support for cerebral functions. Microbiom in the gut has great role
in increasing unbound, free estrogen levels via their β-glucuronidase activity [102,103]. It
is a plausible possibility that gut microbiom colonized in the brain increases the level of
accessible free estrogen.
Adipose tissue is an essential source of estrogen production in extragonadal sites
in both women and men [104]. The functional activity of adipose tissue is regulated by
circulating and locally synthesized estrogens. In the fatty tissue, estrogens are acting in an
autocrine manner, while in the adjacent organs; they increase ER activation in a paracrine
manner [105]. Estrogens are the chief regulators of the health of adipose tissue through
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metabolic and epigenetic pathways [106]. Estrogen exerts its special effects on estrogen
responsive adipocytes by estrogen receptors (E-alpha, ER-beta and GPR30) [107].
In the gonads, the essential precursors of estrogen synthesis are C19 steroids, while
extragonadal sites are unable to synthesize estrogens directly from these factors. With
ageing, increasing estrogen synthesis in peripheral tissues requires a precursor supply from
external sources, for example, dehydroepiandrosterone (DHEA) intake is important [108].
The remarkable volume of ubiquitous fatty tissue and its noteworthy estrogen synthesis justify that fat cells have crucial roles in safeguarding and regulating the signaling
network of neighboring tissues, organs, and the whole body.
Secretory Activities of Visceral Adipose Tissue in Healthy Lean and Obese Cases
Abdominal fatty tissue has crucial secretory functions [109]. Estrogen-regulated genes
orchestrate adipokine, cytokine, and growth factor secretion, which are important signaling
molecules and their estrogen-regulated activation controls the health of the whole body.
Sexual steroids: In adipose tissue, estrogens are the crucial sexual steroids. Appropriate estrogen signaling controls the expression of numerous genes and the coordinated
synthesis of signaling molecules [106].
Adipokines: Leptin controls the equilibrium of energy in the hypothalamus, conferring
anorexinogenic and lipolytic signals. Estrogen treatment results in the increased expression
of leptin receptors in various cells, sensitizing them to leptin [110]. In aromatase knock
out (ARKO) mice with estrogen loss, visceral fat deposition develops and leptin levels
are highly elevated [111]. Adiponectin signaling protects against insulin resistance by
quenching various inflammatory reactions and improving endothelial functions. In adult
mice, oophorectomy increases adiponectin levels, while it may be reduced by estradiol
substitution [112]. Obesity increases the level of resistin, which may be a compensatory
response. In subcutaneous fat cells, an estradiol benzoate treatment decreases resistin
levels [113].
Proinflammatory cytokines and low-grade inflammation: Proinflammatory cytokines
are regulatory proteins which have a great role in the maintenance of genomic and metabolic
stability. In obese fatty tissue, low-grade inflammatory reactions and abundantly expressed cytokines are counteractions to genomic deregulation via increasing estrogen
synthesis [114]. The insulin resistance of obese estrogen deficient adipose tissue leads to
further regulatory disorders in the adjacent organs, resulting in serious co-morbidities,
such as fatty degeneration and malignancies [115,116].
In the low-grade inflammation of obese adipose tissue, increased levels of inflammatory cytokines and immune cell infiltration comprising macrophages and T cells may be
found [117]. Proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α)
and interleukin-6 (IL-6) generate an increased expression and activation of the aromatase
enzyme, resulting in increased estrogen synthesis [118]. Proinflammatory cytokines have
beneficial effects against obesity and obesity-related metabolic disorders via increasing the
aromatase activity and estrogen synthesis. Estrogen treatment of obese ovariectomized
mice decreased the expression of inflammatory cytokines, including TNFα and upregulated estrogen signaling, which improved the insulin sensitivity in both adipose tissue and
liver [119].
Insulin-IGF system. The insulin-like growth factor (IGF) system has a great role in
the regulation and control of growth and differentiation. The receptors of insulin and
insulin-like growth factors work as ligand-specific modulators, regulating various genes
on similar pathway [120]. In the early stage of insulin resistance, an increased IGF-1 level
confers increased insulin synthesis, leading to compensatory hyperinsulinemia.
Harmonized crosstalk and interaction among signaling pathways of ERs and growth
factor receptors (IGF-1R, EGFR, VGFR) are identified in both health and disease [121,122].
In health, growth factor-activated ERs may either facilitate or silence cell growth and
proliferation. In tumors with regulatory defects, abundant growth factor receptors activate
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ERs via unliganded pathway so as to initiate DNA stabilization and apoptotic death rather
than providing excessive proliferative stimulus.
In adipocytes, estrogens control the synthesis of insulin-like growth factor 1 (IGF-1)
and the expression of its receptor (IGF-1R). In turn, the upregulation of IGF-1 synthesis
and its receptor expression increases the unliganded activation of ERs via the AKT and
MAPK regulatory pathways [123]. In an estrogen deficient milieu, increased IGF-1 receptor
signaling stimulates the unliganded activation of ERs, which may momentarily ensure
the genome wide expression of estrogen-regulated genes [64]. In conclusion, in insulin
resistance and obesity, the increased activation and expression of IGF-1 receptors do not
exert pro-oncogenic effects, but rather facilitate unliganded ER activation.
Interaction between adipocytes and immune cells. Adipocytes are in signaling
crosstalk with immune cells in both healthy and obese adipose tissue. In lean adipose
tissue, IL-4 secreted by eosinophil granulocytes and regulatory T (Treg) cells activate M2
type macrophages, which express arginase and anti-inflammatory cytokines such as IL-10.
In contrast, in obese adipose tissue, a high number of M1 type macrophages and increased
secretion of pro-inflammatory cytokines, such as TNFα and IL-6, are coupled with a decrease in anti-inflammatory immune cells [117]. In animal experiments, estrogen is capable
of improving metabolic disorders and, at the same time, exerts anti-inflammatory effects. In
female mice, estrogen protects from adipocyte hypertrophy, obesity, and prevents adipose
tissue oxidative stress and inflammation [124].
In obesity, the upregulation of estrogen signaling restores insulin sensitivity, reduces
lipid deposition, decreases pro-inflammatory cytokine synthesis and quenches inflammatory infiltration. Estrogen treatment provides quite new ways for the prevention and cure
of obesity and obesity-related complications.
6. The Tumor Cell Itself Is the Frontline of Anticancer Combat
According to global medical concepts, tumor cells are enemies to be killed as they
presumably fight for their survival, similar to how pathogenic bacteria fight against antibiotics. Seemingly, tumor cells express cancer driver genes via somatic mutation, and their
altered protein products defeat both the immune defense of body and the therapeutic effect
of pharmaceutical agents.
In reality, the recognition of DNA damage means an emergency state even for tumor
cells. The upregulation of estrogen signaling via the liganded and/or unliganded pathway
is the appropriate means for the restoration of DNA stability. However, in tumors, the
possibility for DNA repair is questionable, attributed to the genomic damage. The more
differentiated a tumor, the stronger its capacity for the compensatory upregulation of
estrogen signaling, coupled with DNA restorative efforts [125].
The spontaneous healing of early breast tumors is a well-known finding justifying
the capacity of initial cancers for self-directed remission. A systematic review and metaanalysis study evaluated a high prevalence of incidental breast cancer and precursor
lesions in autopsy studies on clinically tumor-free cases. The estimated mean prevalence of
incidental cancer and precursor lesions were surprisingly high: 19.5% and 0.85% [126].
Breast cancer is regarded as a multifactorial and very heterogeneous disease that refers
to the abnormal proliferation of the lobular and ductal epithelium of the breast, resulting in
tumor formation [127]. The classifications of breast cancers follow the recommendations of
the World Health Organization (WHO), which are regularly revised in accordance with the
scientific progress [128].
The most important parameter for the classification of breast cancers is their molecular
profile as it was described in 2000 [129]. The heterogeneity of breast cancers at a molecular
level was revealed through the various expression of a panel of genes. Breast cancers
were divided into four main groups: 1. Luminal A (60% of cases); 2. Luminal B (10% of
cases); 3. The overexpression of human epidermal growth factor receptor2 (HER2) (20%
of cases); and 4. Basal-like triple-negative breast cancers (TNBCs) (about 10% of breast
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cancers). Another subgroup has also been described as a normal breast-like subcategory
which resembles the luminal A group but shows a worse prognosis.
In clinical practice, these tumor groups are identified by immunohistochemical markers, such as ER-alpha, progesterone (PR), and human epidermal growth factor receptor
(HER2) expression [127]. In breast cancers, the overexpression of certain receptor families
is mistakenly regarded as an aggressive survival technique and their targeted inhibition is
the principle of current therapeutic measures. In reality, missing or decreased expression of
certain receptors in tumor cells highlights the points of genomic defects requiring repair.
Conversely, the overexpression of certain receptors and regulators, as well as the activating
mutation of their genes indicate the efforts for self-directed genomic repair of tumors
rather than developing survival techniques [12,56]. In reality, the loss of certain receptors
indicates the genomic damage, while the overexpression of others represents the genome
repairing effort.
Immunohistochemical markers of breast cancers show the alterations in their gene
and receptor protein expression as compared to healthy breast epithelium. Molecular
alterations reflecting the grade of DNA damage and the concomitant DNA repairing
actions in different breast cancer subtypes are shown in Table 1.
Table 1. Receptor pattern in breast cancer subtypes reflecting the grade of DNA damage and the
concomitant actions for DNA repair.
Subtype of
Breast Cancer
Receptor
Status
Signs of
DNA Damage
Sigs of
DNA Repair
Proliferative
Activity Endocrine
Response to
Therapy
Luminal A type
(50–60%)
ER overexpression
PR positive
no
ER overexpression
low
good in 50%
Luminal B type
(10%)
ER positive
PR pos/neg
HER2 pos/neg
PR negative
PR positive
HER2 positive
ER positive
increased
moderate/inverse
HER2 enriched
(20%)
ER negative
PR negative
HER2 rich
ER negative
PR negative
HER2 rich
high
no
Triple negative
(10%)
ER negative
PR negative
HER2 negative
ER negative
PR negative
HER2 negative
no
high
no
Luminal type A cancers are the least aggressive tumors with the expression of ER
alpha, and PR. Increased ER expression in breast tumors is traditionally regarded as a
crucial inducer and promoter of tumor growth [127]. This concept derives from confusing
the constellation with causation. Increased ER expression is not a causal factor for tumor
growth, but rather it is an effort for improving estrogen signaling and DNA stabilization in
an estrogen deficient milieu [43].
Estrogen receptor expression was shown to be parallel with DNA repair capacity
in breast cancer cells [130]. This correlation justifies that the high ER expression of untreated tumors is the key to self-directed DNA repair, rather than a fuel for tumor growth.
The strong belief in estrogen induced cancer does not allow consideration of opposite
alternatives.
Luminal A breast cancer may exhibit a transiently good response in 50% of tumors to
adjuvant endocrine therapy; however, near all patients previously showing good tumor
responses later become non-responders [131]. Patients with early luminal ER-positive
breast cancer are at a continuous risk of relapse even after more than 10 years of tamoxifen
treatment [132]. These experiences underline that endocrine disruptor therapy is not
appropriate method even for early ER-positive breast cancer care.
Luminal B tumors are more aggressive than luminal A types. They express lower
ER alpha and lower PR expression or may be PR-negative, in correlation with the weak-
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ening estrogen signal [133]. Luminal B tumors are associated with an increased rate of
p53 mutations and in certain B type tumors, HER2 may also be expressed [134]. Activating
p53 mutations are not oncogenic changes, but rather they mean stronger DNA protection
in tumors with weakening genome stability. In luminal B type tumors, the appearance of
HER2 expression works on the compensatory unliganded activation of ERs [17].
After tamoxifen therapy, patients with ER-positive, PR-negative, and HER2-positive
tumors exhibited higher rates of tumor recurrence and mortality as compared to those
who did not receive the agent [135]. This observation suggests that in type B tumors, the
weakening ER signal is further worsened by endocrine disruptor treatment. In contrast,
Premarin treatment of ER-positive, PR-negative breast cancer cases resulted in a significant
reduction in tumor size and improved patients’ survival [24].
HER2-enriched breast cancer is ER- and PR-negative and HER2-positive. HER-2enriched cancers tend to grow faster than luminal cancers and can have a worse prognosis.
ER- and PR-negativity in HER-2 enriched breast cancers reflects a loss of estrogen signaling
and strong defects in all genomic processes. HER2 overexpression in hormone receptor
negative tumors is mistakenly regarded as a trigger for tumor proliferation, similarly to all
other growth factors [127]. In contrast, in the emergency situation of DNA damage, HER-2
overexpression is a compensatory effort for the unliganded activation of ERs occurring
scarcely in this tumor type [17]. HER-2 protein-targeted therapies against HER-2-enriched
tumors show similarly ambiguous results, like ER-inhibitor anti-estrogens against ERpositive tumors [12].
Triple-negative or basal-like breast cancer is ER-negative, progesterone receptornegative, and HER-2-negative. Triple-negative breast cancer is more common in people
with BRCA1 gene mutation, younger women, and black women. Triple-negative breast
cancers are more aggressive than either luminal A or luminal B breast cancers and they are
not responsive to endocrine therapy [127].
In triple negative breast cancers (TNBCs), the lack of ER, PR, and HER-2 receptors
indicate the serious deregulation of the whole genomic machinery. These tumors are poorly
differentiated and clinically show rapid growth and spread. In TNBC type tumors, there is
no possibility for self-directed DNA repair as ERs seem to be absent or hidden and the regulatory pathways for both liganded and non-liganded ER activations are unnoticeable [43].
The increased risk for TNBC-type tumors in African American women may be attributed
to their excessive pigmentation in a relatively light-deficient geographical region. Poor
light exposure leads to metabolic and hormonal alterations, conferring an increased cancer
risk [136].
The molecular classification of breast cancer types reflects the fact that in women,
stronger estrogen signaling may suppress, while a defective estrogen signal liberates breast
cancer initiation and growth [43]. In tumor cells, the higher the ER expression, the stronger
is the apoptotic effect of therapeutic estrogen exposure. In contrast, endocrine disruptor
therapies may achieve only transient tumor responses in appropriately ER-positive breast
cancers. Poorly differentiated ER/PR-negative and TNBC-type tumors are refractory to
anti-estrogen therapy, attributed to their serious genomic deregulation.
In conclusion, breast cancers are not multifaceted tumors with quite different etiology
and pathogenesis. Consequently, they do not need quite different therapies depending on
their receptor status. The levels of regulatory defects create a line of variously differentiated
tumors between strongly ER-positive, highly differentiated, and poorly differentiated
TNBC-type ones. In breast cancer therapy, natural estrogen is a risk-free available option
for ER-positive tumors [24]. Against ER-negative and TNBC-type poorly differentiated
tumors, Maloney’s mRNA technology would be a promising therapy to be introduced in
the near future [125].
7. Peritumoral Microenvironment: The Second Line of the Antitumor Battle
In the early 2000s, the role of the tumor microenvironment emerged as being an
important player in cancer development, tumor invasion, and metastatic spread [137].
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Today, cancer is regarded as a complex disease built up from the neoplastic lump and
its altered cellular and stromal microenvironment [138,139]. There is a strengthening
belief that tumors insidiously influence all players in their microenvironment via dynamic
intercellular communication. Tumors presumably ensure their invasive growth via escape
from defensive immune reactions and anti-cancer treatment.
The supposed conspiration between tumors and their microenvironment is based on
the belief that all signaling molecules and regulatory proteins are taken for pro-oncogenic
factors when their expression is highly elevated in tumors and in the adjacent cellular
infiltration [139–141]. In addition, when important regulatory genes, such as ESR1, are
accumulated or mutated in tumors, they are regarded as pro-oncogenic alterations, rather
than self-regulated efforts in the repair of genomic damages [142–146]. According to the
reigning preconception, in tumor cells, the upregulation of estrogen signaling and its
activator pathways are regarded as the keys to tumor growth.
In reality, in tumors, the upregulation of certain signaling pathways and activating mutations are not pro-oncogenic factors, but rather they are efforts for metabolic improvement
and genomic stabilization [56]. Unfortunately, advanced tumors have weakened capacities
for self-directed genomic repair and they ask for help via sending messages to their microenvironment. In turn, peritumoral-activated cells send signals and regulatory molecules,
helping the tumor to achieve DNA repair and to commit apoptosis as a kamikaze action.
The re-evaluation of studies on the biochemical and genomic communication between
tumors and activated microenvironmental cells revealed that all signal messages and
transported exosomes aim for the upregulation of each other’s estrogen signaling and the
improvement of all genomic functions. These activating processes serve the elimination of
the tumor rather than helping its proliferation and invasion. In conclusion, the dynamic
communication between the tumor and its microenvironment is a marvelous collaboration
among molecular players fighting for the genomic repair and apoptosis of tumor by means
of their genomic plasticity.
Cancer-associated fibroblasts (CAFs) are major components emerging in the tumor microenvironment. Their assembly and activation may be attributed to signals deriving from
cancer cells [138]. CAFs are in continuous signal communication with cancer cells and all
other cell types in the tumor microenvironment [139]. Distant intercellular communication
occurs by spherical extracellular vesicles (EVs) comprising exosomes carrying different
molecules, such as proteins, DNAs, non-coding RNAs, miRNAs, and mRNAs. Biochemical
and genetic cross-talk between cancer cells and CAFs are important observations; however,
the presumed cooperation for tumor invasion and metastatic spread is not justified, it is a
biased labeling.
Activation of growth factor signaling cascades. In CAFs, the expression of growth
factors, such as the insulin-like growth factor (IGF-1), fibroblast growth factor FGF-7, FGF10, HGF, and TGF-beta 2 are regarded as pro-tumorigenic factors [147]. In reality, estrogen
receptors and growth factor receptors are common regulators of crucial cellular functions
including cell growth and apoptosis, as well as metabolic processes even in tumors [66].
Transforming the growth factor beta (TGF-beta) superfamily is the main inducer
of CAF activation and in turn, CAFs secrete large amount of TGF-beta isoforms for improving tumor cell regulation [148]. Tumor cell-derived extracellular vesicles (EVs) may
frequently contain growth factor TGF-beta, which is regarded as a typical mitogen factor
of tumors [149]. Considering the ER-activating role of growth factors, tumors send them
to CAFs for the activation of their estrogen signal. Tumor-derived EVs, containing certain
miRNAs, contribute to the enhanced TGF-beta expression in CAFs through the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)
signaling pathway [150]. PI3K and AKT/mTOR pathways upregulate ER activation and
improve glucose uptake, which are not pro-tumorigenic processes, but rather increase
anti-tumor activity. Cancer cell-derived EVs, containing mRNA coding for CXCR-4 and
IGF-1R, provoke CAFs for growth factor secretion in acute myeloid leukemia [151].
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Cytokines secreted by CAFs, macrophages and immune cells are important regulators
of inflammatory processes and immune reactions in the tumor microenvironment [152]. Estrogen signaling orchestrates the secretion of both pro-inflammatory and anti-inflammatory
cytokines according to the momentary requirements. Pro-inflammatory cytokines stimulate aromatase activity, estrogen synthesis and ER expression in the estrogen responsive
peritumoral cellular infiltration. When estrogen concentration reaches an appropriately
high concentration, the accumulation of anti-inflammatory cytokines will quench the
inflammatory reaction parallel with the decreasing estrogen level [114].
IL-1β accumulation in hyperplastic lesions activates CAF formation from fibroblasts
via the NF-κB pathway [153], which is a coactivator of ERs, promoting genome stabilization.
Proinflammatory cytokines, IL-6 and TNF-α, are capable of aromatase activation, leading to
increased estrogen concentration and the upregulation of estrogen signaling [154]. In gastric
cancer, tumors send miRNA containing vesicles to CAFs so as to induce inflammatory
cytokine/chemokine secretion through the Janus kinase (JAK)/STAT and NF-κB signaling
pathways [155]. In colorectal cancers, the constitutive mutation of KRAS increases the
activation of EGFR kinase cascades PI3K-Akt and RAS-RAF-MAPK, whereas increases RASGEF signaling pathway, which is related to abundant cytokine production [156]. In Hodgkin
lymphoma, CAFs exposed to tumor cell-derived EVs show increased proinflammatory
cytokine secretion [157]. CAFs activated by tumor EVs, may in turn shed additional EVs
that will transfer signaling and regulatory molecules to tumor cells.
Various tumors promote aromatase activity and estradiol synthesis in the peritumoral stroma via the promotion of proinflammatory cytokine secretion [158]. In breast
cancers, aromatase is abundantly expressed in tumor cells, intratumoral fibrous cells, and
neighboring adipocytes, justifying their collaboration in promotion of excessive estrogen
synthesis [159]. These observations mistakenly support the role of increased estrogen
concentration in tumor growth and invasion.
In contrast, a combined genetic and clinical investigation justified the anti-cancer capacity of increased local estrogen synthesis in tumors and their stroma. In a large prospective
study, the examination of the surgical breast tumor samples revealed a significant correlation between a low aromatase level and an increased loco-regional recurrence rate of
tumors [160]. This study suggests that missing estrogen synthesis in tumors is associated
with worse prognosis in breast cancer cases.
Circulating estradiol may be systemic modulator of CAF secretome as CAFs express
steroid receptors [161]. Estradiol regulates the expression of several microRNAs in CAFs
deriving from breast cancer [162]. In gastric cancer, estrogens stimulate IL-6 secretion
of CAFs, promoting the signal transducer and activator of transcription (STAT-3) expression [163]. The increased expression of STAT3 in CAFs secretome confers an effort for
genome stabilization, as STAT3 is a transcription factor which has an important role in
DNA replication.
Few studies evaluated growth factors and cytokines as positive regulators of the
genome rather than pro-tumorigenic factors. TGF-beta was considered as a tumor suppressor factor due to its cytostatic effect on cancer cells [164]. IL-11 was known for its capacity
to stimulate platelet production in cancer patients with thrombocytopenia [165].
Immune cells in the tumor microenvironment show intense interactions with tumor
cells. The interaction between immune cells and other cell types are regulated by cell surface
immune checkpoints [138].Mast cells are recruited near tumors during tumorgenesis and
release a variety of cytokines and chemokines [166]. Cytokines and chemokines are crucial
regulators of both genomic and immunologic processes and their accumulation is an anticancer effort. Natural killer cells (NK) are cytotoxic and secrete tumor necrosis factor so as
to kill tumor cells [167].
Tumor-associated macrophages (TAMs) infiltrate the microenvironment of tumors and
are mainly divided into two categories: classically activated macrophages (M1 type) and
alternatively activated macrophages (M2 type). The activated M2 type macrophages are
blamed for managing the immune escape of tumors. The abundance of TAM infiltration
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in tumors is mechanically linked with poor disease prognosis [168]. TAM activation and
accumulation in tumors is not a pro-oncogenic feature, but rather their intensive cytokine
secretion is helping aromatase activity and increasing estrogen concentration.
Myeloid-derived suppressor cells (MDSC) have apparently immunosuppressive effects; they may block immunotherapy and may play a role in tumor maintenance and
progression [169]. MDSCs also accumulate in response to the chronic inflammation and
lipid deposition in obesity and contribute to the more rapid progression of cancers in
obese individuals. In reality, the accumulation of MDSCs is not a causal factor of rapid
tumor progression and obesity associated inflammation, but rather it seems to be an intense
immune defense against metabolic disorder associated tumors.
Tumor-infiltrating lymphocytes (TILs) are important participants of the tumor microenvironment [152]. Immune cell infiltrates may exhibit ambiguous properties, either
promoting or inhibiting tumor progression depending on the features of the primary tumor [170]. CD4+ T cell polarization has been identified as a mediator of tumor immune
surveillance. T helper 1 (Th1) cell functions are associated with tumor suppression and the
upregulation of IFNγ and IL-12. T helper 2 (Th2) responses are reliant on IL-4 production
and presumably exhibit tumor-promoting activity [171,172]. Murine and human studies
reported that increased E2 concentration induces increased Th2 responses and upregulates
IL-4 secretion [173,174].
A remarkable fact is that constellation of strong estrogen signal and increasing tumor
growth does not justify causal correlation. A recent study reported increased immune
cell infiltrate comprising Th1 T cells, B cells, and cytotoxic T lymphocytes (CTLs) in ERnegative breast tumors as compared to ER-positive cancers [175]. The correlation between
ER-negative breast tumors and more intensive immune cell infiltration strongly suggests
that poorly differentiated tumors with a loss of estrogen signaling need stronger immune
support for their DNA repair than highly differentiated ER-positive ones.
Gene expression analysis in ER-positive breast cancer patients showed that blocking
the liganded ER activation with aromatase inhibitor (letrozole) continuously increased
the tumor infiltration with B cell and T helper lymphocyte subsets following treatment
initiation [158]. This result justified that letrozole inhibition of estrogen signal in ER-positive
tumors induced an emergency state, promptly recruiting strong immune cell infiltration.
In conclusion, tumors and their microenvironment are allies in the fight against worsening genomic defects and consequential tumor invasion. The more serious the genomic
damage of a tumor, the denser is the peritumoral immune cell infiltration attributed to
the emergency state. Invasive tumor spread, coupled with intensive peritumoral cellular
infiltration, may be regarded as a common failure of tumor and peritumoral cells rather
than the victory of presumably conspirator partners.
8. Molecular Changes in Tumors Responsive and Non-Responsive to Endocrine Therapy
The traditional belief of estrogen-induced breast cancer required the introduction
of inhibitors of estrogen signaling for breast cancer care. The pharmaceutical industry
developed two kinds of anti-estrogens for therapeutic purposes: a selective estrogen
receptor modulator—tamoxifen—and an aromatase inhibitor (AI)—letrozole [176]. Since
the early 1970s, anti-estrogens are commonly used compounds for breast cancer care as
adjuvant therapy.
In breast cancer cases, anti-estrogen therapy caused many difficulties from the onset because of the development of so-called endocrine resistance in tumors. Results of
anti-estrogen use could not surpass the “magic” 30% of tumor response rate, showing
similar weaknesses to other endocrine therapies like oophorectomy or high dose synthetic
estrogen [177]. About 70% of overall breast cancers could not respond to anti-estrogen
therapy, showing stagnation or an even faster growth. Moreover, about half of the targeted
ER-positive breast cancers exhibited primary resistance to anti-estrogen treatment [131].
Moreover, near all patients showing earlier good tumor responses to endocrine treat-
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ment later experienced secondary resistance, leading to metastatic disease and a fatal
outcome [178].
In the past decades, great efforts were exerted for revealing the mechanism of presumed endocrine resistance of ER-positive breast cancers so as to predict responses to
adjuvant endocrine therapy in patients. Researchers mistakenly supposed that both responsive and non-responsive tumor cells are aggressive enemies, developing various techniques
in fighting for their survival [12].
8.1. Successful Fight of Anti-estrogen Responsive Tumors against the Endocrine
Disruptor Treatment
In tumors responsive to anti-estrogen, the chief action against AF2 blockade is the
restoration and amplification of the estrogen activation of ERs [56]:
1. Tamoxifen treatment provokes compensatory unliganded ER activation without
delay by ER-alpha translocation from the nucleus to the membrane-bound EGFRs [179]
(Figure 1); 2. The long term “therapeutic” ER blockade amplifies the expression of the
ER-alpha coactivator; AIB1 (amplified in breast cancer 1) [180]. Under tamoxifen treatment,
another coactivator of ERs, cyclin D1 amplifies the activation of both STAT3 and ERs [181];
3. Tamoxifen treatment highly activates the transcription factor NFκB and its upregulative
interaction with ER-alpha [182,183]; 4. Tamoxifen induces the increasing expression of
certain microRNAs that bind to ER mRNAs, activating the translational processes [184];
5. Tamoxifen provokes the amplification of the ESR1 gene associated with the increased
expression and activation of ERs [185,186] (Figure 2); 6. Aromatase inhibitor treatment
provokes an acquired amplification of the CYP19A1 gene, increasing both aromatase
expression and estrogen synthesis [187]; 7. In tumor cells treated with tamoxifen, abundant
lncRNA transcripts of ERs mediate the activating mutations for crucial genes of the genome
stabilizer circuit; such as ESR1, BRCA1, and CYP19A [56].
Figure
1. Rapid
response
to Tamoxifen
(T) induced
ER ER
blockade
in cancer
cells.The
rapid
translocaFigure
1. Rapid
response
to Tamoxifen
(T) induced
blockade
in cancer
cells.The
rapid
translocation
tion of
unbound
estrogen
receptors
(ERs)
out
of
the
nucleus
helps
their
interactions
with
membraneof unbound estrogen receptors (ERs) out of the nucleus helps their interactions with membraneassociated growth factor receptors; GFRs (IGF1-R, EGFR). Cytoplasmic ERs activated by growth
associated growth factor receptors; GFRs (IGF1-R, EGFR). Cytoplasmic ERs activated by growth
factor receptors initiate rapid transcriptional processes in the nucleus via transcriptional factors
factor receptors initiate rapid transcriptional processes in the nucleus via transcriptional factors (TFs).
(TFs). Growth factor (GF)-activated GFRs may also induce unliganded activation on nuclear unGrowth
factor (GF)-activated
GFRs may
also induce
unliganded
activation on nuclear
unbound
bound
ERs, driving
their transcriptional
activity.
E: estrogen,
P: phosphorylation,
N: nucleus,
Dot- ERs,
driving
their
transcriptional
activity.
E:
estrogen,
P:
phosphorylation,
N:
nucleus,
Dotted
arrow:
ted arrow: activation, black solid arrow: inhibition, red arrow: schematic DNA segment.
activation, black solid arrow: inhibition, red arrow: schematic DNA segment.
Cancers 2024, 16, 1573
associated growth factor receptors; GFRs (IGF1-R, EGFR). Cytoplasmic ERs activated by growth
factor receptors initiate rapid transcriptional processes in the nucleus via transcriptional factors
(TFs). Growth factor (GF)-activated GFRs may also induce unliganded activation on nuclear unbound ERs, driving their transcriptional activity. E: estrogen, P: phosphorylation, N: nucleus, Dotted arrow: activation, black solid arrow: inhibition, red arrow: schematic DNA segment. 18 of 28
Figure
2. Molecular
mechanism
ofoftumor
Tamoxifen(T)
(T)treated
treated
cancer
cells.
Increased
Figure
2. Molecular
mechanism
tumorresponse
response in
in Tamoxifen
cancer
cells.
Increased
estradiol
(E
2
)
concentration
activates
newly
expressed
abundant
estrogen
receptors
(ERs)
increasing
estradiol (E2 ) concentration activates newly expressed abundant estrogen receptors (ERs) increasing
the expression
of estrogen-regulated
growthfactors
factors
(GFs)
activate
growth
the expression
of estrogen-regulatedgenes.
genes.In
Inthe
the meantime,
meantime, growth
(GFs)
activate
growth
factorfactor
receptors
(GFRs)
conferring
unliganded
forfree
freenuclear
nuclear
ERs.
The
predominance
receptors
(GFRs)
conferring
unliganded activation
activation for
ERs.
The
predominance
of of
estradiol
(E2) (E
bound
ERs
over
T
bound
ones
leads
to
DNA
repair,
apoptotic
death
and
clinical
tumor
estradiol
)
bound
ERs
over
T
bound
ones
leads
to
DNA
repair,
apoptotic
death
and
clinical
tumor
2
response.
P: phosphorylation,
Dottedarrow:
arrow:
activation,
solid arrow:
inhibition,
response.
P: phosphorylation,N:
N:nucleus,
nucleus, Dotted
activation,
blackblack
solid arrow:
inhibition,
red
red arrow:
schematic
DNA
segment.
arrow: schematic DNA segment.
8.2. Unsuccessful Fight of Tumors Non Responsive to Endocrine Disruptor Treatment
In anti-estrogen responsive breast cancers, the increased regulatory processes promote the compensatory improvement of estrogen activation of ERs and may achieve a
successful tumor response [188]. Earlier anti-estrogen responsive breast cancers become
non-responsive as the possibilities for liganded ER activation are exhausted. In nonresponsive tumors, increased growth factor receptor signaling remains an ultimate refuge
for unliganded ER activation and DNA stabilization [17]. However, when the liganded ER
activation is completely blocked, the increased unliganded activation of ERs is incapable of
restoring ER signaling (Figure 3).
In anti-estrogen resistant breast cancers, physiological regulatory pathways are working so as to increase unliganded ER activation. In tamoxifen-resistant cancers, the ER
coactivator HOXB7 exhibits an increased expression and may activate kinase phosphorylation of both EGFR [189] and HER2 [190], promoting unliganded ER activation. Further
ER coactivators—AIB1 and HER2/neu—stimulate hormone-free ER activation [191]. In
tumor xenografts, both ER and HER2 activations were coupled with the compensatory
activation of MUCIN4 [192]. In anti-estrogen resistant tumors, the increased expressions of
plasma membrane-bound EGFRs [193] and IGF-1Rs [194,195] amplify unliganded ER activation. In endocrine-resistant cancers, acquired somatic mutations may strongly increase
the compensatory hormone-free ER activation.
Cancers 2024, 16, 1573
mor xenografts, both ER and HER2 activations were coupled with the compensatory activation of MUCIN4 [192]. In anti-estrogen resistant tumors, the increased expressions of
plasma membrane-bound EGFRs [193] and IGF-1Rs [194,195] amplify unliganded ER activation. In endocrine-resistant cancers, acquired somatic mutations may strongly increase
19 of 28
the compensatory hormone-free ER activation.
Figure
3. Molecular
mechanism
of tumor
resistance
in Tamoxifen
(T)-treated
cancer
cells.cells.
The The
Figure
3. Molecular
mechanism
of tumor
resistance
in Tamoxifen
(T)-treated
cancer
liganded
activation
of
abundant
nuclear
estrogen
receptors
(ERs)
is
completely
blocked
by
T-bindliganded activation of abundant nuclear estrogen receptors (ERs) is completely blocked by T-binding.
ing. The compensatory abundant expression of membrane-associated growth factor receptors
The compensatory abundant expression of membrane-associated growth factor receptors (GFRs)
(GFRs) struggles for the unliganded activation of T-bound ERs. However, the T blockade inhibits
struggles for the unliganded activation of T-bound ERs. However, the T blockade inhibits the
the restoration of ER signaling resulting in unrestrained proliferation. GF: growth factor, N: nucleus,
ofinhibition,
ER signaling
resulting
in unrestrained
proliferation.
GF: growth
factor,
N: nucleus,
blackrestoration
solid arrow:
spiral:
unsuccessful
activation,
red arrow: schematic
DNA
segment.
black solid arrow: inhibition, spiral: unsuccessful activation, red arrow: schematic DNA segment.
In tumors resistant to endocrine therapy, acquired somatic mutations may strongly
In tumors resistant to endocrine therapy, acquired somatic mutations may strongly
increase the compensatory hormone-free activation of ERs:
increase the compensatory hormone-free activation of ERs:
1. Estrogen conferred somatic mutation of ERBB2 gene amplifies the expression and
1. Estrogen conferred somatic mutation of ERBB2 gene amplifies the expression and
activity of growth factor receptors, conferring estrogen-free ER activation [191]; 2. In enactivity of growth factor receptors, conferring estrogen-free ER activation [191]; 2. In
docrine refractory ER-positive breast tumors, the PIK3CA gene is frequently mutated, upendocrine refractory ER-positive breast tumors, the PIK3CA gene is frequently mutated,
regulating the components of the PI3K-AKT-mTOR pathway and increasing hormone free
upregulating the components of the PI3K-AKT-mTOR pathway and increasing hormone
free ER activation [196]; 3. In AI-resistant breast cancers, acquired point mutations in the
ligand binding domain (LBD) of ESR1 gene confer hormone-independent activation of
ERs [142]; 4. In anti-estrogen resistant tumors, chromosomal rearrangement on the ESR1
gene leads to somatic mutations driving an increased unliganded activation of ERs [144];
5. In tamoxifen-resistant tumor cells, the activation of the PI3K/AKT pathway led to
a significant increase in BARD1 and BRCA1 protein expressions via increased estrogen
independent activation of ERs [197].
9. Estrogen Induced Apoptosis Is Promising in Both the Prevention and Therapy
of Cancer
Estrogen treatment of breast cancers resistant to either long term estrogen deprivation
(LTED-R) or tamoxifen (TAM-R) triggers an apoptotic death in tumors [198].
In clinical practice, estrogen dramatically decreased the mortality of advanced breast
cancer cases after stopping the long term tamoxifen therapy [199]. Following long term
estrogen deprivation, estrogen reduced metastatic tumors and prolonged the survival of
patients [200]. The biology of estrogen-induced apoptosis in breast and prostatic cancers
seem to be promising in both the prevention and therapy of tumors [201].
Breast cancers unresponsive to anti-estrogen treatment exhibit extreme upregulation of both ER and GFR expressions. Estrogen may exert intensive anti-cancer capacity
via balanced liganded and unliganded activation of abundant ERs. In reality, estrogen
treatment does not return non-responsive tumors to anti-estrogen sensitivity. Conversely,
Cancers 2024, 16, 1573
20 of 28
estrogen helps tumor cells to defeat the genotoxic drug as they are highly sensitized to
estrogen signal.
Important lessons may be drawn from the 50 years of breast cancer therapy with
anti-estrogens: 1. In tumors, there is no endocrine therapy resistance, but rather the
possibilities for compensatory ER activation are exhausted; 2. In tumors responsive to
anti-estrogen therapy, increased ER expression and activation is not a survival technique,
but rather it is an effort for increasing estrogen signaling; 3. In tumors non-responsive to
anti-estrogen therapy, increased growth factor receptor expression and activation is not a
survival technique, but rather it is an effort for compensatory unliganded ER activation; 4.
Tumors exhaustively treated by aromatase inhibitors, show genomic plasticity, exhibiting
acquired mutations on the ligand binding domain of ESR1 gene conferring new, hormoneindependent activation of modified ERs in the absence of estrogen.
10. Conclusions
Compared to various organs, female breasts exhibit unique sensitivity to genomic
instability caused by either germline or acquired gene mutations. This fact may partially
explain why breast cancer has become the flagship of cancer research. Although the preconception of “estrogen-induced” breast cancer has led breast cancer care to a quite erroneous
pathway, a thorough examination of the controversies between estrogen signaling and
cancer development yielded valuable progress in overall cancer research.
The correlation between genomic instability and conspicuously increased breast cancer
risk in germline BRCA gene mutation carriers revealed that the defect in the genome
stabilizer circuit is the origin of cancer initiation, rather than excessive estrogen signaling.
Defects in ER, BRCA, or the aromatase enzyme upsets the triangular partnership of these
regulatory proteins, leading to weaknesses in estrogen signaling and genomic instability.
BRCA mutation carrier healthy and tumor cells similarly show efforts for increasing the
liganded and unliganded ER activation and for compensatory upregulation of another
genome safeguarding protein, p53.
Understanding the fight of cancer cells for the activation of estrogen signaling, together
with genome stabilization, reveals the secret of various receptor landscapes of breast cancer
subtypes. In tumors, the increased expression of hormone receptors reflects efforts for
increasing liganded ER activation, while the overexpression of HER2 represents trying
to increase unliganded ER activation. The blockade of either ERs or HER2s seems to be
an erroneous therapeutic concept. Breast cancers are not resistant to genotoxic therapies,
but rather they exhausted all possibilities for defending the remnants of genomic stability.
Progressive genomic instability leads to unrestrained proliferative activity.
The cellular infiltration of the tumor microenvironment is not an organic part of tumors.
Inflammatory cells are recruited by the tumor itself and the intercellular communication by
messages and extracellular vesicles confer in asking for help. The stronger the genomic
deregulation in the tumor, the denser is the adjacent infiltration of activated mesenchymal
and immune competent cells. Immune competent cells do not need therapeutic genomic
machination as they know exactly their task in the anti-cancer fight. When tumor invasion is coupled with dense peritumoral infiltration, supportive genome repairing therapy
is necessary, rather than the disruption of mutation-activated DNA repair pathways of
tumors.
In conclusion, the improvement of genomic stability may be the new strategy in cancer
therapy. The upregulation of estrogen signaling leads to strengthened immune response,
whilst inducing the apoptotic death of tumors in a Janus-faced manner.
Funding: This research received no external funding.
Conflicts of Interest: The author declares no conflicts of interest.
Cancers 2024, 16, 1573
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