Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL African-American (A... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL African-American (AA) breast cancer patients have lower survival compared with Caucasian-American (CA) patients. This survival disparity is significant in view of the lower incidence of breast cancer among AA. Lower incidence of breast cancer is associated with parity and lactation in young women. Lactation's protective effect is mediated by fatty-acid binding protein 3 (FABP3), a protein that promotes differentiation of breast epithelial cells. We have demonstrated that AA normal women below 50 years have lower FABP3 levels as compared to CA women (p = 0.030). FABP3 is a protective tumor suppressor that increases during pregnancy and lactation, in response to the lactation hormone prolactin (PRL). PRL effects on the breast are mediated by IGF-II. Thus, we propose that PRL treatment of normal epithelial breast cell lines would increase IGF-II to promote differentiation, increase FABP3, and decrease FABP5. FABP5 is associated with metastasis and may compensate for FABP3 decrease. We treated MCF10A (CA) and AG11132 (AA) normal breast epithelial cell lines with recombinant human PRL (0-200 ng/ml) for 24 hours. Cell lysates were examined for protein and mRNA levels of IGF-II, FABP3, and FABP5. In MCF10A cells, PRL stimulated an increase in protein levels of IGF-II (p=0.044), and a decrease in protein levels of FABP5 (p=0.044). Protein levels of FABP3 significantly decreased with increasing PRL concentration (p=0.030). IGF-II mRNA levels also increased at 50 and 200 ng/ml PRL (2.59 and 2.58 fold, respectively). In AG11132 cells, protein levels of FABP3 significantly increased with increasing PRL concentration (p=0.016). IGF-II and FABP5 mRNA levels increased significantly at 10 ng/ml PRL (49.91 and 2.13 fold, respectively); however, no corresponding changes were seen in protein levels. Though both cell lines respond to PRL treatment, only the MCF10A line displays increased IGF-II and decreased FABP5, which are protective changes in normal mammary cells. FABP3 levels decreased in MCF10A cells at higher PRL concentrations; this may be due to untreated MCF10A cells expressing significantly more FABP3 and prolactin, relative to AG11132 cells. The higher levels of PRL and FABP3 in untreated MCF10A cells indicate that they are more differentiated and thus require less PRL stimulation, relative to the AG11132 cells, to achieve a differentiated state. The dissimilar responsiveness of these cell lines to PRL may suggest decreased protection against breast carcinogenesis in AA patients, and may help us to further understand this health disparity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 22. doi:1538-7445.AM2012-22
Cancer Epidemiology and Prevention Biomarkers, Nov 1, 2007
A76 Recognition that racial differences may account for a disparity in survival among African Ame... more A76 Recognition that racial differences may account for a disparity in survival among African American (AA) women with breast cancer has stimulated nationwide interest in research to elucidate the contributing factors. The increased mortality rate for AA women is paradoxical in view of their lower risk of breast cancer (BC) incidence. Thus, a comprehensive approach to understand the biological factors associated with poorer outcomes among AA patients is urgently needed. We are addressing this need by investigating how IGF-II contributes to the survival disparity observed among AA breast cancer patients. IGF-II is a fetal growth factor regulated by estrogen to promote proliferation and prevent apoptosis. Expression of this growth factor in human breast cancer cells stimulated rapid tumor growth in a nude mice model of human BC developed in our laboratory. Of great significance, the expression of IGF-II abrogated the requirement of estrogen for tumor growth and metastasis in this animal model. These observations led us to propose that IGF-II plays a key role in BC by promoting estrogen-independent growth, a hallmark of aggressive BC characteristic of tumors observed among AA patients. How does IGF-II promotes estrogen independent growth? What is the interaction between estrogen signaling and IGF signaling? Estrogens play a key role in the progression of human breast cancer. The effects of estrogen are largely mediated by two different, but related, estrogen receptor (ER) isoforms named estrogen receptor alpha (ER- α) and estrogen receptor beta (ER- β). While both, ER- αand ER- β, share a number of structural and functional similarities, they differ in term of subcellular localization and transactivation of certain estrogen responsive genes. On the other hand, IGF-II binds the IGF-1 receptor, a membrane associated tyrosine kinase that initiates a downstream signaling cascade to stimulate growth. In spite of distinct signaling cascades, recent published work revealed that the estrogen-IGF signaling pathways interact in a“cross-talk” where the activation of the IGF-IR can phosphorylate and activate the ER in cell membranes to stimulate a rapid non-genomic response. This activation of the estrogen receptor does not require estrogen. These observations have prompted us to investigate whether IGF-II alters the phosphorylation and sub-cellular localization of these ERs. Breast cancer cells from Caucasian and AA breast cancer patients were obtained from ATCC. Cells were sonicated and fractionated to separate the distinct compartments (cytosol, organelles, nucleus and extracellular matrix). Phosphorylated and non-phosphorylated ER- α and ER- β were analyzed by Western blot. Densitometric analysis was used to quantify the differences detected in the bands revealed by chemiluminescence in x-ray films. Our study revealed that phosphorylated ER- α and ER- β were detected in membranes, but not nucleus, of cells classified as ER-α. Cells from AA patients expressed higher amounts of ER- α in the plasma membrane while ER- β was mainly detected in the mitochondria. We propose that IGF-II mediates estrogen actions in these cells by phosphorylating/activating both receptors to promote estrogen independent growth and control mitochondrial function to provide energy and chemoresistance, thus contributing to the survival disparity observed among AA breast cancer patients.
Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Gr... more Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Overexpression of HER2 is linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity. Dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the insulin-like growth factor 1 receptor (IGF1R), the insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). We hypothesize that IGF2 mediates trastuzumab-resistance in JIMT-1 cell line. IGF2 mRNA and protein levels were determined in trastuzumab-sensitive cell lines (BT474, SKBR3, and ZR 75-30) and trastuzumab-resistant cell line (JIMT-1) by qPCR and Western blot. We also analyzed the effect of IGF2 inhibition treatment on the phosphorylation levels of HER2. Our study showed that IGF2 increases in trastuzumab-resistant cell line, JIMT-1. Furthermore, inhibition of IGF2 decreases JIMT-1 cell viability compared to control and renders cells sensitive to trastuzumab. These findings provide a potential therapy target for trastuzumab-resistant breast cancers. Citation Format: Xousaen M. Helu, Daisy D. De Leon. IGF2 role in trastuzumab-resistance in JIMT-1 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2013-538
Publisher Summary This chapter provides an overview of insulin-like growth factor-binding protein... more Publisher Summary This chapter provides an overview of insulin-like growth factor-binding proteins (IGF-BP). The insulin-like growth factors (IGFs)—or somatomedins—constitute a family of growth hormone (GH)-dependent peptides with both anabolic and mitogenic activities for a wide variety of tissues and cell lines. IGF-I and -II are characterized by a striking structural homology with human proinsulin. The human gene for IGF-II is located on the short arm of chromosome 11 in close proximity to the gene for insulin and spans over 30 kb of DNA. This gene is composed of at least eight exons. The preprohormone consists of a 24-amino-acid signal peptide, a 67-amino-acid mature peptide, and a carboxy-terminal peptide of 89 amino acids. Significant diversity exists in the 5'-untranslated regions, where different mRNA species arise as a result of distinct promoters and alternative RNA splicing. Both IGF-I and -II are synthesized in a wide variety of tissues. The close relationship between insulin and IGF-I receptors is not surprising, given the binding affinities and structural similarities of the two receptors. Unlike the insulin and IGF-I receptors, the type 2 IGF receptor is characterized by a long extracellular domain, containing 15 repeat sequences of approximately 150 residues, a 23-residue transmembrane domain, and a small 164-residue cytoplasmic domain. No homology exists between the type 2 IGF receptor and the insulin or type 1 receptor. The initial characterization of IGF-BPs was done by gel filtration chromatography. The chapter discusses radioreceptor assays, affinity labeling, western ligand blotting, and immune-precipitation among other methods of detection.
The Journal of Clinical Endocrinology and Metabolism, 1995
Insulin-like growth factor-binding protein-4 (IGFBP-4) is an important regulator of insulin-like ... more Insulin-like growth factor-binding protein-4 (IGFBP-4) is an important regulator of insulin-like growth factor-I (IGF-I) anabolic activity in bone. Although cultured human osteoblast-like (hOB) cells have been reported to secrete IGFBP-4, we could not detect IGFBP-4 protein in 8 of 27 individual donor-derived hOB-cell conditioned medium (hOB-CM) samples examined by Western ligand blotting. Nonetheless, this subset of hOB cells had normal IGFBP-4 messenger ribonucleic acid expression and protein secretion. Regulation of IGFBP-4 levels in hOB cultures appeared to occur extracellularly. hOB cells produce an IGFBP-4 proteinase that requires the presence of IGF for cleavage of the IGFBP-4 molecule into 2 fragments of approximately 18 and 14 kilodaltons. These fragments are not detected by Western ligand blotting. Our data indicate that elevated endogenous levels of IGF can activate IGFBP-4 proteolysis, because in hOB cultures lacking detectable IGFBP-4 protein 1) basal IGF messenger ribonucleic acid expression was increased; 2) IGF-II peptide levels were elevated; 3) IGF-neutralizing antibodies added to hOB-CM attenuated the proteolysis of exogenous IGFBP-4; and 4) recombinant human IGFBP-4 was proteolyzed into 2 immunoreactive fragments of approximately 18 and 14 kilodaltons during cell-free incubations in these hOB-CM without the addition of exogenous IGF. In conclusion, elevated IGF expression and secretion can contribute to enhanced proteolysis of endogenous and exogenous IGFBP-4 via a proteinase secreted by cultured hOB cells. Levels of endogenous IGF peptide may determine IGFBP-4 availability in the bone microenvironment and, thus, modulate the local cell response to IGF-I.
ABSTRACT The present study was designed to compare the expression of the Jun family of protooncog... more ABSTRACT The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using 32P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c-jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 days and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7-fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA-protein complex was reduced by c-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that c-jun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection.
African-American (AA) women with breast cancer are more likely to have advanced disease at diagno... more African-American (AA) women with breast cancer are more likely to have advanced disease at diagnosis, higher risk of recurrence and poorer prognosis than Caucasian (CA) females. Insulin-like growth factor- II (IGF-II) is a potent mitogen that induces cell proliferation and survival signals through activation of the IGF-I (IGF-IR), insulin (INSR) and hybrid receptors. We hypothesize that differential expression of the IGF1R and INSR isoforms (IR-A, IR-B) between AA and CA women potentiates IGF-II mitogenic effects, thus contributing to the health disparity observed between these ethnic groups. We examined IGF-II, IGF1R and INSR isoforms protein expression and phosphorylation in paired breast tissue samples from AA and CA women by western blot analysis, immunohistochemistry and ELISA techniques. IGF-II expression was significantly higher in AA cell lines and tissue samples when compared to Caucasians. Furthermore, significantly increased expression of IGF1R was observed in AA normal tissues as compared to CA normal tissues, while IGF1R expression was similar between AA normal and malignant tissues. Moreover, AA tumor samples expressed significantly lower levels of IR-B protein than CA tumor samples. IGF1R, INSR, IRS-1 and Shc phosphorylation were significantly higher in AA tumor samples. We conclude that the differential expression and phosphorylation of IGF-II, IGF1R and INSR between AA and CA females coupled with differential activation of downstream signaling pathways may contribute to the increased risk of malignant transformation seen in young AA females, and the more aggressive breast cancer phenotype observed among AA breast cancer patients and represent, along with IGF-II, potential therapeutic targets against health disparities in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 281.
African-American (AA) patients present with more advanced breast cancer and have lower survival a... more African-American (AA) patients present with more advanced breast cancer and have lower survival at all ages compared with Caucasian-Americans (CA). While AA women have a lower overall breast cancer incidence than CA women, AA women have a higher mortality rate. These differences may be due in part to socioeconomic factors; however emerging evidence suggests that biological factors may also play a role in this health disparity. Altered fatty acid binding protein (FABP) expression has been demonstrated in breast cancer. FABP3 is a protective tumor suppressor that has been shown to inhibit breast cell proliferation, and is downregulated in breast cancer tissues. Levels of FABP3 correspond with mammary differentiation, and reach their height during pregnancy and lactation, in response to very high prolactin expression. Thus, FABP3 is correlated with the protective effect against breast cancer induced by lactation. FABP5, another member of the FABP family, is associated with metastasis and is upregulated in breast cancer tissues. Hence, increased levels of FABP5 may compensate for decreased levels of FABP3. Since FABP3 is associated with decreased breast cell proliferation, and FABP5 is associated with increased proliferation, we reasoned that differential expression of these proteins in breast tissues would correlate with breast cancer incidence and progression. Therefore, we hypothesized that higher incidence of breast cancer is associated with lower FABP3 and consequently, higher FABP5. We analyzed protein and mRNA levels of the FABPs in paired breast tissue samples from AA and CA women by Western blot analysis and RT-PCR. Data was divided into two groups- ages below 50, and ages 50 and above. Protein measurements showed that the ratio of FABP3 to FABP5 levels (FABP ratio) was significantly higher in AA patients below 50 compared to those 50 and older (p=0.059) and compared to age-matched CA patients (p=0.049). FABP3 mRNA levels were significantly higher in AA malignant (AAM) tissues from ages below 50 (p=0.003), and significantly lower in AAM tissues from ages 50 and older (p<0.001), relative to CA malignant (CAM) counterparts. FABP5 mRNA levels were significantly higher in AAM tissues from ages 50 and older (p=0.023), relative to CAM counterparts. These findings, particularly in the older age group, demonstrate that differential FABP expression may contribute to the survival disparity between AA and CA breast cancer patients. Our studies provide much needed information about the mechanisms involved in the protective effect lactation induces in the breast and will potentially offer new tools for preventive breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5605. doi:10.1158/1538-7445.AM2011-5605
Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Gr... more Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Increased levels of HER2 are linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity and dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor(IGF1R) in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the IGF1R, insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). Previous findings in our lab demonstrate an increased expression of IGF2 in trastuzumab-resistance cell line JIMT1 when compared to trastuzumab-sensitive cell lines BT474, SKBR3, and ZR75-30. We hypothesis that to overcome trastuzumab-resistance in JIMT1 cell lines, dual inhibition of HER2 and IGF2 is necessary to induce cell death. When JIMT1 cells were treated with resveratrol, both HER2 and IGF2 mRNA and protein levels were reduced and a decrease in cell viability was observed. We also analyzed the phosphorylation levels of HER2 and IGF1R. Our studies suggest that blocking both HER2 and IGF2 is necessary to decrease cell viability in trastuzumab-resistant JIMT1 cells. Citation Format: Xousaen M. Helu, Daisy De Leon. Inhibition of HER2 and IGF2 triggers cell death in trastuzumab-resistant HER2 positive JIMT1 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2014-4244
Triple-negative breast cancer (TNBC) is very aggressive, resistant to chemotherapy and more likel... more Triple-negative breast cancer (TNBC) is very aggressive, resistant to chemotherapy and more likely to relapse, causing the worst prognosis. African American (AA) women suffer higher incidence and mortality of TNBC due to the expression of high levels of Insulin Growth Factor 2 (IGF2) which promotes tumor progression, metastasis, and chemoresistance. Also, it has been established that functional mitochondria and mitochondrial DNA (mtDNA) are essential for cancer cell growth. Mutations and/or reductions in mtDNA copy number that alter the Oxidative Phosphorylation (OXPHOS) physiology are common features of TNBC. We have demonstrated that mtDNA content is lower in CRL-2335 AA TNBC cell line when compared to the CRL-2335 IGF2 knockout cell line. Thus, we propose that IGF2 regulates the mtDNA content. This study was designed to demonstrate if IGF2 regulates mitochondrial genes to determine the cell energy phenotype. An XFp analyzer was used to study the mitochondrial function in terms of OCR (Oxygen Consumption Rate/Mitochondrial Respiration) and ECAR (Extracellular Acidification Rate/ Glycolysis) in the wild type and IGF2 stable knockout of CRL-2335 AA TNBC cells. Real Time PCR was performed to study the gene expression pattern of IGF2, PGC1α and PGC1β. PGC1α and PGC1β are critical genes in the regulation of the mitochondrial biogenesis, thus, they are important in the cellular metabolic phenotype. Utilizing the Seahorse metabolic system we assessed cell energy phenotype and alterations in terms of mitochondrial respiration rate (OCR) % and Glycolysis (ECAR) %. Our preliminary results showed that the overall OCR and ECAR of the stressed CRL-2335 AA TNBC cells was altered according to the levels of IGF-II expressed. Furthermore, the results demonstrated that there was a metabolic shift in the CRL-2335 AA TNBC cells towards the glycolytic pathway when IGF2 was knockout in comparison to the wild type CRL-2335 cells. Also, IGF2 knockout cells showed higher gene expression rate of PGC1β as compared to the wild type. The above data confirms that IGF2 plays a critical role in determining the cell energy phenotype. Citation Format: Maria L. Pagan, Vinodh Kumar Radhakrishnan, Daisy De Leon. IGF2 regulates mitochondrial cell energy phenotype and biogenesis in TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2017-4421
Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL African-American (A... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL African-American (AA) breast cancer patients have lower survival compared with Caucasian-American (CA) patients. This survival disparity is significant in view of the lower incidence of breast cancer among AA. Lower incidence of breast cancer is associated with parity and lactation in young women. Lactation's protective effect is mediated by fatty-acid binding protein 3 (FABP3), a protein that promotes differentiation of breast epithelial cells. We have demonstrated that AA normal women below 50 years have lower FABP3 levels as compared to CA women (p = 0.030). FABP3 is a protective tumor suppressor that increases during pregnancy and lactation, in response to the lactation hormone prolactin (PRL). PRL effects on the breast are mediated by IGF-II. Thus, we propose that PRL treatment of normal epithelial breast cell lines would increase IGF-II to promote differentiation, increase FABP3, and decrease FABP5. FABP5 is associated with metastasis and may compensate for FABP3 decrease. We treated MCF10A (CA) and AG11132 (AA) normal breast epithelial cell lines with recombinant human PRL (0-200 ng/ml) for 24 hours. Cell lysates were examined for protein and mRNA levels of IGF-II, FABP3, and FABP5. In MCF10A cells, PRL stimulated an increase in protein levels of IGF-II (p=0.044), and a decrease in protein levels of FABP5 (p=0.044). Protein levels of FABP3 significantly decreased with increasing PRL concentration (p=0.030). IGF-II mRNA levels also increased at 50 and 200 ng/ml PRL (2.59 and 2.58 fold, respectively). In AG11132 cells, protein levels of FABP3 significantly increased with increasing PRL concentration (p=0.016). IGF-II and FABP5 mRNA levels increased significantly at 10 ng/ml PRL (49.91 and 2.13 fold, respectively); however, no corresponding changes were seen in protein levels. Though both cell lines respond to PRL treatment, only the MCF10A line displays increased IGF-II and decreased FABP5, which are protective changes in normal mammary cells. FABP3 levels decreased in MCF10A cells at higher PRL concentrations; this may be due to untreated MCF10A cells expressing significantly more FABP3 and prolactin, relative to AG11132 cells. The higher levels of PRL and FABP3 in untreated MCF10A cells indicate that they are more differentiated and thus require less PRL stimulation, relative to the AG11132 cells, to achieve a differentiated state. The dissimilar responsiveness of these cell lines to PRL may suggest decreased protection against breast carcinogenesis in AA patients, and may help us to further understand this health disparity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 22. doi:1538-7445.AM2012-22
Cancer Epidemiology and Prevention Biomarkers, Nov 1, 2007
A76 Recognition that racial differences may account for a disparity in survival among African Ame... more A76 Recognition that racial differences may account for a disparity in survival among African American (AA) women with breast cancer has stimulated nationwide interest in research to elucidate the contributing factors. The increased mortality rate for AA women is paradoxical in view of their lower risk of breast cancer (BC) incidence. Thus, a comprehensive approach to understand the biological factors associated with poorer outcomes among AA patients is urgently needed. We are addressing this need by investigating how IGF-II contributes to the survival disparity observed among AA breast cancer patients. IGF-II is a fetal growth factor regulated by estrogen to promote proliferation and prevent apoptosis. Expression of this growth factor in human breast cancer cells stimulated rapid tumor growth in a nude mice model of human BC developed in our laboratory. Of great significance, the expression of IGF-II abrogated the requirement of estrogen for tumor growth and metastasis in this animal model. These observations led us to propose that IGF-II plays a key role in BC by promoting estrogen-independent growth, a hallmark of aggressive BC characteristic of tumors observed among AA patients. How does IGF-II promotes estrogen independent growth? What is the interaction between estrogen signaling and IGF signaling? Estrogens play a key role in the progression of human breast cancer. The effects of estrogen are largely mediated by two different, but related, estrogen receptor (ER) isoforms named estrogen receptor alpha (ER- α) and estrogen receptor beta (ER- β). While both, ER- αand ER- β, share a number of structural and functional similarities, they differ in term of subcellular localization and transactivation of certain estrogen responsive genes. On the other hand, IGF-II binds the IGF-1 receptor, a membrane associated tyrosine kinase that initiates a downstream signaling cascade to stimulate growth. In spite of distinct signaling cascades, recent published work revealed that the estrogen-IGF signaling pathways interact in a“cross-talk” where the activation of the IGF-IR can phosphorylate and activate the ER in cell membranes to stimulate a rapid non-genomic response. This activation of the estrogen receptor does not require estrogen. These observations have prompted us to investigate whether IGF-II alters the phosphorylation and sub-cellular localization of these ERs. Breast cancer cells from Caucasian and AA breast cancer patients were obtained from ATCC. Cells were sonicated and fractionated to separate the distinct compartments (cytosol, organelles, nucleus and extracellular matrix). Phosphorylated and non-phosphorylated ER- α and ER- β were analyzed by Western blot. Densitometric analysis was used to quantify the differences detected in the bands revealed by chemiluminescence in x-ray films. Our study revealed that phosphorylated ER- α and ER- β were detected in membranes, but not nucleus, of cells classified as ER-α. Cells from AA patients expressed higher amounts of ER- α in the plasma membrane while ER- β was mainly detected in the mitochondria. We propose that IGF-II mediates estrogen actions in these cells by phosphorylating/activating both receptors to promote estrogen independent growth and control mitochondrial function to provide energy and chemoresistance, thus contributing to the survival disparity observed among AA breast cancer patients.
Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Gr... more Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Overexpression of HER2 is linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity. Dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the insulin-like growth factor 1 receptor (IGF1R), the insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). We hypothesize that IGF2 mediates trastuzumab-resistance in JIMT-1 cell line. IGF2 mRNA and protein levels were determined in trastuzumab-sensitive cell lines (BT474, SKBR3, and ZR 75-30) and trastuzumab-resistant cell line (JIMT-1) by qPCR and Western blot. We also analyzed the effect of IGF2 inhibition treatment on the phosphorylation levels of HER2. Our study showed that IGF2 increases in trastuzumab-resistant cell line, JIMT-1. Furthermore, inhibition of IGF2 decreases JIMT-1 cell viability compared to control and renders cells sensitive to trastuzumab. These findings provide a potential therapy target for trastuzumab-resistant breast cancers. Citation Format: Xousaen M. Helu, Daisy D. De Leon. IGF2 role in trastuzumab-resistance in JIMT-1 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2013-538
Publisher Summary This chapter provides an overview of insulin-like growth factor-binding protein... more Publisher Summary This chapter provides an overview of insulin-like growth factor-binding proteins (IGF-BP). The insulin-like growth factors (IGFs)—or somatomedins—constitute a family of growth hormone (GH)-dependent peptides with both anabolic and mitogenic activities for a wide variety of tissues and cell lines. IGF-I and -II are characterized by a striking structural homology with human proinsulin. The human gene for IGF-II is located on the short arm of chromosome 11 in close proximity to the gene for insulin and spans over 30 kb of DNA. This gene is composed of at least eight exons. The preprohormone consists of a 24-amino-acid signal peptide, a 67-amino-acid mature peptide, and a carboxy-terminal peptide of 89 amino acids. Significant diversity exists in the 5'-untranslated regions, where different mRNA species arise as a result of distinct promoters and alternative RNA splicing. Both IGF-I and -II are synthesized in a wide variety of tissues. The close relationship between insulin and IGF-I receptors is not surprising, given the binding affinities and structural similarities of the two receptors. Unlike the insulin and IGF-I receptors, the type 2 IGF receptor is characterized by a long extracellular domain, containing 15 repeat sequences of approximately 150 residues, a 23-residue transmembrane domain, and a small 164-residue cytoplasmic domain. No homology exists between the type 2 IGF receptor and the insulin or type 1 receptor. The initial characterization of IGF-BPs was done by gel filtration chromatography. The chapter discusses radioreceptor assays, affinity labeling, western ligand blotting, and immune-precipitation among other methods of detection.
The Journal of Clinical Endocrinology and Metabolism, 1995
Insulin-like growth factor-binding protein-4 (IGFBP-4) is an important regulator of insulin-like ... more Insulin-like growth factor-binding protein-4 (IGFBP-4) is an important regulator of insulin-like growth factor-I (IGF-I) anabolic activity in bone. Although cultured human osteoblast-like (hOB) cells have been reported to secrete IGFBP-4, we could not detect IGFBP-4 protein in 8 of 27 individual donor-derived hOB-cell conditioned medium (hOB-CM) samples examined by Western ligand blotting. Nonetheless, this subset of hOB cells had normal IGFBP-4 messenger ribonucleic acid expression and protein secretion. Regulation of IGFBP-4 levels in hOB cultures appeared to occur extracellularly. hOB cells produce an IGFBP-4 proteinase that requires the presence of IGF for cleavage of the IGFBP-4 molecule into 2 fragments of approximately 18 and 14 kilodaltons. These fragments are not detected by Western ligand blotting. Our data indicate that elevated endogenous levels of IGF can activate IGFBP-4 proteolysis, because in hOB cultures lacking detectable IGFBP-4 protein 1) basal IGF messenger ribonucleic acid expression was increased; 2) IGF-II peptide levels were elevated; 3) IGF-neutralizing antibodies added to hOB-CM attenuated the proteolysis of exogenous IGFBP-4; and 4) recombinant human IGFBP-4 was proteolyzed into 2 immunoreactive fragments of approximately 18 and 14 kilodaltons during cell-free incubations in these hOB-CM without the addition of exogenous IGF. In conclusion, elevated IGF expression and secretion can contribute to enhanced proteolysis of endogenous and exogenous IGFBP-4 via a proteinase secreted by cultured hOB cells. Levels of endogenous IGF peptide may determine IGFBP-4 availability in the bone microenvironment and, thus, modulate the local cell response to IGF-I.
ABSTRACT The present study was designed to compare the expression of the Jun family of protooncog... more ABSTRACT The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using 32P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c-jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 days and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7-fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA-protein complex was reduced by c-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that c-jun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection.
African-American (AA) women with breast cancer are more likely to have advanced disease at diagno... more African-American (AA) women with breast cancer are more likely to have advanced disease at diagnosis, higher risk of recurrence and poorer prognosis than Caucasian (CA) females. Insulin-like growth factor- II (IGF-II) is a potent mitogen that induces cell proliferation and survival signals through activation of the IGF-I (IGF-IR), insulin (INSR) and hybrid receptors. We hypothesize that differential expression of the IGF1R and INSR isoforms (IR-A, IR-B) between AA and CA women potentiates IGF-II mitogenic effects, thus contributing to the health disparity observed between these ethnic groups. We examined IGF-II, IGF1R and INSR isoforms protein expression and phosphorylation in paired breast tissue samples from AA and CA women by western blot analysis, immunohistochemistry and ELISA techniques. IGF-II expression was significantly higher in AA cell lines and tissue samples when compared to Caucasians. Furthermore, significantly increased expression of IGF1R was observed in AA normal tissues as compared to CA normal tissues, while IGF1R expression was similar between AA normal and malignant tissues. Moreover, AA tumor samples expressed significantly lower levels of IR-B protein than CA tumor samples. IGF1R, INSR, IRS-1 and Shc phosphorylation were significantly higher in AA tumor samples. We conclude that the differential expression and phosphorylation of IGF-II, IGF1R and INSR between AA and CA females coupled with differential activation of downstream signaling pathways may contribute to the increased risk of malignant transformation seen in young AA females, and the more aggressive breast cancer phenotype observed among AA breast cancer patients and represent, along with IGF-II, potential therapeutic targets against health disparities in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 281.
African-American (AA) patients present with more advanced breast cancer and have lower survival a... more African-American (AA) patients present with more advanced breast cancer and have lower survival at all ages compared with Caucasian-Americans (CA). While AA women have a lower overall breast cancer incidence than CA women, AA women have a higher mortality rate. These differences may be due in part to socioeconomic factors; however emerging evidence suggests that biological factors may also play a role in this health disparity. Altered fatty acid binding protein (FABP) expression has been demonstrated in breast cancer. FABP3 is a protective tumor suppressor that has been shown to inhibit breast cell proliferation, and is downregulated in breast cancer tissues. Levels of FABP3 correspond with mammary differentiation, and reach their height during pregnancy and lactation, in response to very high prolactin expression. Thus, FABP3 is correlated with the protective effect against breast cancer induced by lactation. FABP5, another member of the FABP family, is associated with metastasis and is upregulated in breast cancer tissues. Hence, increased levels of FABP5 may compensate for decreased levels of FABP3. Since FABP3 is associated with decreased breast cell proliferation, and FABP5 is associated with increased proliferation, we reasoned that differential expression of these proteins in breast tissues would correlate with breast cancer incidence and progression. Therefore, we hypothesized that higher incidence of breast cancer is associated with lower FABP3 and consequently, higher FABP5. We analyzed protein and mRNA levels of the FABPs in paired breast tissue samples from AA and CA women by Western blot analysis and RT-PCR. Data was divided into two groups- ages below 50, and ages 50 and above. Protein measurements showed that the ratio of FABP3 to FABP5 levels (FABP ratio) was significantly higher in AA patients below 50 compared to those 50 and older (p=0.059) and compared to age-matched CA patients (p=0.049). FABP3 mRNA levels were significantly higher in AA malignant (AAM) tissues from ages below 50 (p=0.003), and significantly lower in AAM tissues from ages 50 and older (p<0.001), relative to CA malignant (CAM) counterparts. FABP5 mRNA levels were significantly higher in AAM tissues from ages 50 and older (p=0.023), relative to CAM counterparts. These findings, particularly in the older age group, demonstrate that differential FABP expression may contribute to the survival disparity between AA and CA breast cancer patients. Our studies provide much needed information about the mechanisms involved in the protective effect lactation induces in the breast and will potentially offer new tools for preventive breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5605. doi:10.1158/1538-7445.AM2011-5605
Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Gr... more Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Increased levels of HER2 are linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity and dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor(IGF1R) in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the IGF1R, insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). Previous findings in our lab demonstrate an increased expression of IGF2 in trastuzumab-resistance cell line JIMT1 when compared to trastuzumab-sensitive cell lines BT474, SKBR3, and ZR75-30. We hypothesis that to overcome trastuzumab-resistance in JIMT1 cell lines, dual inhibition of HER2 and IGF2 is necessary to induce cell death. When JIMT1 cells were treated with resveratrol, both HER2 and IGF2 mRNA and protein levels were reduced and a decrease in cell viability was observed. We also analyzed the phosphorylation levels of HER2 and IGF1R. Our studies suggest that blocking both HER2 and IGF2 is necessary to decrease cell viability in trastuzumab-resistant JIMT1 cells. Citation Format: Xousaen M. Helu, Daisy De Leon. Inhibition of HER2 and IGF2 triggers cell death in trastuzumab-resistant HER2 positive JIMT1 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2014-4244
Triple-negative breast cancer (TNBC) is very aggressive, resistant to chemotherapy and more likel... more Triple-negative breast cancer (TNBC) is very aggressive, resistant to chemotherapy and more likely to relapse, causing the worst prognosis. African American (AA) women suffer higher incidence and mortality of TNBC due to the expression of high levels of Insulin Growth Factor 2 (IGF2) which promotes tumor progression, metastasis, and chemoresistance. Also, it has been established that functional mitochondria and mitochondrial DNA (mtDNA) are essential for cancer cell growth. Mutations and/or reductions in mtDNA copy number that alter the Oxidative Phosphorylation (OXPHOS) physiology are common features of TNBC. We have demonstrated that mtDNA content is lower in CRL-2335 AA TNBC cell line when compared to the CRL-2335 IGF2 knockout cell line. Thus, we propose that IGF2 regulates the mtDNA content. This study was designed to demonstrate if IGF2 regulates mitochondrial genes to determine the cell energy phenotype. An XFp analyzer was used to study the mitochondrial function in terms of OCR (Oxygen Consumption Rate/Mitochondrial Respiration) and ECAR (Extracellular Acidification Rate/ Glycolysis) in the wild type and IGF2 stable knockout of CRL-2335 AA TNBC cells. Real Time PCR was performed to study the gene expression pattern of IGF2, PGC1α and PGC1β. PGC1α and PGC1β are critical genes in the regulation of the mitochondrial biogenesis, thus, they are important in the cellular metabolic phenotype. Utilizing the Seahorse metabolic system we assessed cell energy phenotype and alterations in terms of mitochondrial respiration rate (OCR) % and Glycolysis (ECAR) %. Our preliminary results showed that the overall OCR and ECAR of the stressed CRL-2335 AA TNBC cells was altered according to the levels of IGF-II expressed. Furthermore, the results demonstrated that there was a metabolic shift in the CRL-2335 AA TNBC cells towards the glycolytic pathway when IGF2 was knockout in comparison to the wild type CRL-2335 cells. Also, IGF2 knockout cells showed higher gene expression rate of PGC1β as compared to the wild type. The above data confirms that IGF2 plays a critical role in determining the cell energy phenotype. Citation Format: Maria L. Pagan, Vinodh Kumar Radhakrishnan, Daisy De Leon. IGF2 regulates mitochondrial cell energy phenotype and biogenesis in TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2017-4421
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