ORIGINAL RESEARCH ARTICLE
published: 23 April 2014
doi: 10.3389/fonc.2014.00087
Protein Kinase C beta in the tumor microenvironment
promotes mammary tumorigenesis
Julie A. Wallace 1,2 † , Jason R. Pitarresi 1,2 † , Nandini Sharma 1,2 , Marilly Palettas 1,2 , Maria C. Cuitiño 2 ,
Steven T. Sizemore 2,3 , Lianbo Yu 2,4 , Allen Sanderlin 1,2 , Thomas J. Rosol 2,5 , Kamal D. Mehta 1 ,
Gina M. Sizemore 1,2 * and Michael C. Ostrowski 1,2 *
1
2
3
4
5
Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH, USA
Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA
Department of Radiation Oncology, The Ohio State University, Columbus, OH, USA
Center for Biostatistics, The Ohio State University, Columbus, OH, USA
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA
Edited by:
Jozsef Dudas, Medical University
Innsbruck, Austria
Reviewed by:
Alexandre Arcaro, University of Bern,
Switzerland
Silvia Pastorekova, Slovak Academy of
Sciences, Slovakia
*Correspondence:
Gina M. Sizemore, Department of
Molecular and Cellular Biochemistry,
The Comprehensive Cancer Center,
The Ohio State University, 570A
Biomedical Research Tower, 460
West, 12th Avenue, Columbus, OH
43210, USA
e-mail: gina.sizemore@osumc.edu;
Michael C. Ostrowski , Department of
Molecular and Cellular Biochemistry,
The Comprehensive Cancer Center,
The Ohio State University, 598
Biomedical Research Tower, 460
West, 12th Avenue, Columbus, OH
43210, USA
e-mail: michael.ostrowski@
osumc.edu
Protein kinase C beta (PKCβ) expression in breast cancer is associated with a more aggressive tumor phenotype, yet the mechanism for how PKCβ is pro-tumorigenic in this disease
is still unclear. Interestingly, while it is known that PKCβ mediates angiogenesis, immunity,
fibroblast function and adipogenesis, all components of the mammary tumor microenvironment (TME), no study to date has investigated whether stromal PKCβ is functionally
relevant in breast cancer. Herein, we evaluate mouse mammary tumor virus–polyoma
middle T-antigen (MMTV–PyMT ) induced mammary tumorigenesis in the presence and
absence of PKCβ. We utilize two model systems: one where PKCβ is deleted in both the
epithelial and stromal compartments to test the global requirement for PKCβ on tumor
formation, and second, where PKCβ is deleted only in the stromal compartment to test its
role in the TME. MMTV–PyMT mice globally lacking PKCβ live longer and develop smaller
tumors with decreased proliferation and decreased macrophage infiltration. Similarly, when
PKCβ is null exclusively in the stroma, PyMT -driven B6 cells form smaller tumors with
diminished collagen deposition. These experiments reveal for the first time a tumor promoting role for stromal PKCβ in MMTV–PyMT tumorigenesis. In corroboration with these
results, PKCβ mRNA (Prkcb ) is increased in fibroblasts isolated from MMTV–PyMT tumors.
These data were confirmed in a breast cancer patient cohort. Combined these data suggest the continued investigation of PKCβ in the mammary TME is necessary to elucidate
how to effectively target this signaling pathway in breast cancer.
Keywords: protein kinase C beta, breast cancer, mammary neoplasms (experimental), tumor microenvironment,
stroma, fibroblasts
†
Julie A. Wallace and Jason R.
Pitarresi have contributed equally to
this work.
INTRODUCTION
A role for protein kinase C (PKC) in cancer has been known
for over 20 years when it was first recognized that phorbol esters
promoted tumor formation through activation of the PKC family [reviewed in Ref. (1)]. There are 10 isozymes that make up
this family of serine–threonine kinases, which is classified into
three major groups by their mode of activation: the classical
PKCs (PKCα, PKCβI, PKCβII, and PKCγ) are calcium and diacylglycerol (DAG) dependent; the novel PKCs (PKCδ, PKCε, PKCη,
and PKCθ) are activated by DAG; and the atypical PKCs (PKCξ
and PKCι) are independent of calcium and DAG. PKCβ is comprised of two splice variants, PKCβI and PKCβII, which are from
the same gene (PRKCB) and differ only in their last 50 amino
acids (2, 3). Differential PKC expression has been documented
in essentially every histological cancer type including carcinomas
(e.g., breast, colorectal), sarcomas (e.g., glioma), lymphomas (e.g.,
diffuse large B-cell lymphoma), and leukemias (e.g., B-cell chronic
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lymphocytic leukemia) [reviewed in Ref. (1, 4)]. Functionally,
PKCs mediate various physiological processes such as proliferation, differentiation, apoptosis, cellular motility, and angiogenesis
(1, 4).
Relevance for PKC in breast cancer was first reported in 1986
when Borner and colleagues observed that PKC is increased in
more aggressive estrogen receptor-α (ERα) negative mammary
tumor cells compared to their less aggressive ERα positive counterparts (5). This putative tumor promoting role was supported
shortly thereafter by evidence of increased PKC enzymatic activity
in breast tumor versus normal patient samples (6, 7). Evaluation
of the specific PKC isozymes soon followed and PKCα (8–14),
PKCβ (as discussed below), PKCδ (15, 16), PKCε (17), and PKCη
(18, 19) were all found to have roles in breast cancer progression.
Investigation of PKCβ, specifically, has revealed that both splice
variants (PKCβI and PKCβII) are upregulated in breast tumor versus matched normal patient tissue (20), with cytoplasmic PKCβII
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Wallace et al.
associating with Ki-67 expression and HER2 positivity (21). Functionally, ectopic overexpression of either wild type (WT) or constitutively active PKCβI or PKCβII increases breast cancer cell line
growth in vitro through upregulation of cyclin D1, while inhibition of kinase activity decreases growth as seen via introduction
of dominant negative PKCβI or PKCβII, and through treatment
with LY379196, a PKCβ selective inhibitor (22). Interestingly, even
though PKCβII is associated with increased proliferation and
highly aggressive HER2 positive disease, its expression does not
correlate with patient survival in this cohort (21). An independent study also observed that PKCβ mRNA (PRKCB) correlates
with increased grade and triple negative (ERα−/PR−/HER2−)
disease, but not relapse-free survival (23).
This discrepancy between the in vitro breast cancer cell line
and patient survival data may be due to PKCβ-mediating physiological processes within the breast tumor microenvironment
(TME), which could cause the evaluation of PKCβ expression
in whole tumor tissue, which contains both tumor and stroma,
to be misleading. All epithelial-derived tumors (i.e., carcinomas)
are surrounded by a complex TME that contains extracellular
matrix proteins, endothelial cells, immune cells, and fibroblasts
[reviewed in Ref. (24)]. Importantly, PKCβ function in nonepithelial cell types has been described in several model systems.
Within the endothelial compartment of the TME, PKCβ is a wellknown downstream effector of vascular endothelial growth factor
(VEGF) signaling, which mediates angiogenesis (25–27). PKCβ
is also involved in immunity through activation of NF-κB (28–
30). Less is known about PKCβ specifically in tumor-associated
fibroblasts, but it was recently shown that PKC is required for
pancreatic cancer-associated fibroblast (CAF) invasiveness (31)
as well as resistance to irradiation-induced apoptosis in primary
human fibroblasts (32). PKCβ is, however, required for extracellular matrix and collagen deposition in rodent models of diabetes
(33, 34). The breast TME also includes adipocytes and PKCβ null
mice have decreased adipose tissue with altered lipid metabolism (35, 36). Combined, these studies suggest important roles
for PKCβ in the breast TME and the need for further elucidation
of PKCβ’s roles in this context.
Herein, we utilized PKCβ genomic knockout mice (37) to
evaluate mammary tumorigenesis through the use of two model
systems: one where PKCβ is deleted in both the mammary epithelium and the TME, and one where PKCβ is deleted only in the
TME. We reveal for the first time a requirement for PKCβ in
MMTV–PyMT (mouse mammary tumor virus–polyoma middle T-antigen) induced mammary tumor growth. MMTV–PyMT
mice lacking PKCβ (Prkcb −/− ) in both epithelial and stromal compartments have increased tumor latency with a decrease in tumor
load and tumor volume. Decreased tumorigenesis in the MMTV–
PyMT ; Prkcb −/− mice is accompanied by diminished tumor proliferation and macrophage infiltration with angiogenesis seemingly unaffected. To test directly whether PKCβ in the TME is
functionally relevant, we orthotopically injected B6 PyMT tumor
cells, a MMTV–PyMT derived mammary tumor cell line (38), into
WT or Prkcb −/− mice. Tumor volume was similarly decreased
in the absence of stromal PKCβ confirming a requirement for
PKCβ in the mammary cancer TME. Most interestingly, collagen deposition was decreased in this model. Moreover, fibroblasts
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PKCβ promotes mammary tumorigenesis
isolated from the MMTV–PyMT tumors have dramatically higher
levels of PKCβII mRNA (Prkcb) than WT mammary fibroblasts,
whereas Prkcb in the MMTV–PyMT epithelium is decreased. This
increase in stromal PRKCB is similarly observed in a human breast
cancer patient cohort confirming the translational relevance of
PKCβ function in the TME.
MATERIALS AND METHODS
ANIMAL BREEDING
All animal procedures were approved by The Ohio State University Institutional Animal Care and Use Committee (protocol
#2007A0120-R2). Prkcb −/− (35, 37) and MMTV–PyMT mice
(39) have been described. The MMTV–PyMT mice were bred
into C57Bl/6 at least 10 generations. MMTV–PyMT males were
bred with Prkcb +/− females to generate MMTV–PyMT ; Prkcb +/−
male progeny. The MMTV–PyMT ; Prkcb +/− males were then
bred with Prkcb +/− females to generate MMTV–PyMT ; Prkcb +/+
and MMTV–PyMT ; Prkcb −/− female progeny for tumor analysis.
Genotyping primers and conditions for the Prkcb −/− mice have
been previously reported (35). Genotyping primers and conditions for the MMTV–PyMT mice are described on The Jackson
Laboratory website.
TUMOR STUDIES AND FIBROBLAST/EPITHELIAL ISOLATION
MMTV–PyMT ; Prkcb +/+ and MMTV–PyMT ; Prkcb −/− mice
were palpated starting at 3 months of age. Upon detection of palpable tumors, tumorigenesis was allowed to progress three additional
weeks, upon which the tumors were harvested. At the time of harvest, tumor burden was measured as a percentage of the tumor
weight relative to total body weight, and total tumor volume was
calculated by caliper measurements (length × width × height).
Each tumor was then either frozen or fixed in formalin for later histological analysis. For the orthotopic injection study, we injected
3 × 106 B6 PyMT tumor cells into the inguinal mammary fat pads
of either Prkcb +/+ or Prkcb −/− mice. Tumors were monitored by
weekly palpation. When the largest tumor reached 1 cm in diameter, a final measurement was determined by caliper and all tumors
were harvested. B6 PyMT cells were a generous gift from Tsonwin Hai and have been described previously (38). B6 PyMT cells
were maintained in vitro using DMEM/F-12 plus 10% fetal bovine
serum plus penicillin/streptomycin. Isolation of mammary fibroblasts and epithelium was performed by gravity separation as has
been described (40, 41).
IMMUNOFLUORESCENCE AND HISTOLOGICAL STAINING
Tumor tissue was fixed in formalin for 24 h, transferred to 70%
ethanol, paraffin embedded, and sectioned (5 µm). Sections were
rehydrated through xylenes and an ethanol series. Antigen retrieval
was performed using DAKO antigen retrieval in a steamer for
30 min and sections were blocked with DAKO protein block. Primary detection of F4/80 (Molecular Probes), Ki-67 (DAKO), and
MECA-32 (BD Pharmingen), was performed overnight at 4°C.
Alexafluor-488 and Alexafluor-596 (Invitrogen) were used for secondary detection and DAPI as a nuclear counterstain. To quantify,
five images (20× fields) were taken per mouse. Quantification of
F4/80 and Ki-67 staining was done using the count tool in Adobe
Photoshop CS5. Percent positivity of each stain was determined
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relative to the total number of cells as visualized by the DAPI
counterstain. Quantification of MECA-32 staining was measured
as percent positive area using Image J (42). Hematoxylin and eosin
(H&E) and Masson’s Trichrome staining was performed by the
Solid Tumor Pathology Core at OSU.
RNA ISOLATION AND QUANTITATIVE REAL-TIME PCR
Total RNA was isolated using Trizol reagent (Invitrogen), treated
with Turbo DNase I (Ambion) and cDNA generated using Superscript III Reverse Transcriptase (Invitrogen), all per manufacturer’s
recommendations. Prkcb (F = gaaactcgaacgcaaggaga; R = accg
gtcgaagttttcagc; Probe #83) and Rpl4 (F = gatgagctgtatggcacttgg;
R = cttgtgcatgggcaggtta; Probe #38) were detected using the Roche
Universal Probe Library system.
DATASET AND STATISTICAL ANALYSES
The Karnoub breast cDNA microarray dataset of human breast
tumors was retrieved from Oncomine (oncomine.org) for in silico
analyses (43). PRKCB expression was compared between invasive ductal breast carcinoma samples versus normal breast tissue.
Statistical significance was determined by Student’s t -test assuming a two-tailed distribution and equal variance. Kaplan–Meier
FIGURE 1 | Global PKCβ deletion reduces mammary tumorigenesis.
(A) Representative H&E stained mammary tumors from PyMT; Prkcb + / +
and PyMT; Prkcb −/− mice. Scale bar, 100 µm. (B) Kaplan–Meier curve
comparing overall tumor-free survival of PyMT; Prkcb + / + and PyMT; Prkcb −/−
mice (n = 17 per group, *P < 0.02 obtained by log-rank). (C) Prkcb
expression in mammary tumor cells of PyMT; Prkcb + / + and PyMT; Prkcb −/−
mice normalized to Rpl4 (n = 3 and 2, respectively, bars represent
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PKCβ promotes mammary tumorigenesis
survival curves were generated and statistical significance determined using log-rank. Tumor volume measurements and genetic
marker staining was compared between two genetic groups. Log
transformation of the data was performed if needed. Two-sample
t -test or non-parametric Mann–Whitney test was applied after
normality check. Type I error level was controlled at 0.05 level,
and adjusted by Bonferroni’s method when multiple comparisons
were made.
RESULTS
GLOBAL PKCβ DELETION REDUCES MAMMARY TUMORIGENESIS
Members of the PKC family are known to influence proliferation,
differentiation, apoptosis, cellular motility, and angiogenesis (1, 4),
leading us to hypothesize that loss of PKCβ would cause decreased
mammary tumorigenesis in the MMTV–PyMT mammary tumor
model. To evaluate the role for PKCβ in tumorigenesis, we bred the
Prkcb −/− knockout mice with those harboring the MMTV –PyMT
transgene, a well-characterized murine model of mammary tumor
formation (44). We monitored tumor development and harvested tumors 3 weeks after initial palpation. Although the tumors
between the two groups looked histologically similar by hematoxylin and eosin (H&E) staining (Figure 1A), MMTV–PyMT
means + standard deviation). (D) Dissected tumor load (percentage of
tumor mass relative to total body mass) from PyMT; Prkcb + / + and PyMT;
Prkcb −/− mice (n = 16 per group, *P < 0.05 obtained from Mann–Whitney
test). (E) Dissected tumor volume (mm3 ) isolated from PyMT; Prkcb + / + and
PyMT; Prkcb −/− mice (n = 16 and 14, respectively, *P < 0.05 obtained from
two-sample t -test of log based 10 transformation applied on original right
skewed data).
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mice lacking PKCβ (MMTV–PyMT; Prkcb −/− ) developed tumors
at a longer latency when compared to control MMTV–PyMT ;
Prkcb +/+ mice (Figure 1B). The absence of Prkcb in the tumors
was confirmed (Figure 1C). Not only did the absence of PKCβ
delay tumor onset, but the tumors that developed in the MMTV–
PyMT ; Prkcb −/− mice were smaller as measured by both tumor
load (Figure 1D) as well as total volume (Figure 1E). These data
indicate that the global absence of PKCβ in this mouse model has
a measurable impact on mammary tumorigenesis.
GLOBAL PKCβ DELETION DECREASES TUMOR CELL PROLIFERATION
AND MACROPHAGE RECRUITMENT, BUT NOT TUMOR VASCULATURE
To explain the delayed onset and decreased tumor size in mice
lacking PKCβ, we stained for a proliferation marker (Ki-67)
within the PyMT tumors of both genetic groups. We saw an
observable decrease in the percentage of Ki-67 positive cells in
MMTV–PyMT ; Prkcb −/− mice (Figure 2A), indicating that PKCβ
influences PyMT tumor progression by modulating tumor cell
proliferation capacity. Given the recent knowledge that the TME
can influence tumor cell proliferation (41), as well as the known
function for PKCβ in endothelium and immune cells, we also
FIGURE 2 | Global PKCβ deletion decreases tumor cell proliferation and
macrophage recruitment, but not tumor vasculature. (A) Left:
representative images of Ki-67 staining of proliferating cells within PyMT;
Prkcb + / + and PyMT; Prkcb −/− tumors in vivo. Scale bar, 50 µm. Right: graph
of percentage of Ki-67+ cells relative to total DAPI+ cells (n = 3 per group,
bars represent means + standard deviation, *P = 0.074 obtained from
two-sample t -test). (B) Left: representative images of F4/80 staining of
macrophage cells within PyMT; Prkcb + / + and PyMT; Prkcb −/− tumors in vivo.
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PKCβ promotes mammary tumorigenesis
investigated macrophage infiltration and tumor vasculature in the
MMTV–PyMT tumors with and without PKCβ.We saw a significant decrease in percentage of F4/80 positive area, a macrophage
marker, in MMTV–PyMT; Prkcb −/− tumors (Figure 2B), indicating that loss of PKCβ within macrophages influences their recruitment to the tumor site. We saw no measurable difference in tumor
vascularization, as measured by MECA-32 staining, between the
Prkcb −/− and WT groups (Figure 2C), demonstrating that the
delayed tumor onset and growth observed in Prkcb −/− mice was
independent of vasculature formation.
LOSS OF PKCβ IN STROMAL COMPARTMENTS DECREASES TUMOR
VOLUME AND COLLAGEN DEPOSITION, BUT HAS NO EFFECT ON
TUMOR CELL PROLIFERATION, VASCULARIZATION, OR MACROPHAGE
INFILTRATION
To determine the importance of PKCβ signaling within the stroma,
we orthotopically injected Prkcb −/− and WT mice with B6 PyMT derived tumor cells. As seen with the genetic model, tumors
developing in Prkcb −/− mice exhibited no obvious histological differences (Figure 3A), but did result in significantly smaller tumor
volumes compared to WT controls (Figure 3B). This result, in
Scale bar, 50 µm. Right: graph of percentage of F4/80+ area relative to total
area of image (n = 3 per group, bars represent means + standard deviation,
*P < 0.05 obtained from two-sample t -test). (C) Left: representative images
of MECA-32 staining of tumor vasculature within PyMT; Prkcb + / + and PyMT;
Prkcb −/− tumors in vivo. Scale bar, 50 µm. Right: graph of percentage of
MECA-32+ area relative to total area of image (n = 3 per group, bars
represent means + standard deviation, P > 0.05 obtained from two-sample
t -test).
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FIGURE 3 | Loss of PKCβ in stromal compartments decreases tumor
volume. (A) Representative H&E stained B6 PyMT tumors developed in
wild type (WT) and Prkcb −/− mice. Scale bar, 100 µm. (B) Dissected B6
conjunction with the results presented in Figure 1, confirms that
stromal PKCβ is important in PyMT mammary tumor formation.
In order to further assay the effect of stromal PKCβ deletion within
the tumor and its microenvironment, tumors were also evaluated
for histological alterations within the distinct cellular compartments. Interestingly, there was no difference in Ki-67, F4/80, or
MECA-32 positivity between Prkcb −/− and WT mice (Figure 4).
Thus, losing PKCβ signaling in the stroma decreased tumor size,
but had no effect on tumor cell proliferation, vascularization, or
macrophage recruitment. To evaluate whether stromal fibroblasts
could be responsible for the observed decrease in tumor size in
Prkcb −/− mice, we evaluated collagen deposition through Masson’s Trichrome staining. The absence of PKCβ specifically in the
stroma resulted in significantly diminished collagen deposition
while this decrease was not as pronounced in the global knockout model (Figure 5). These results imply that PKCβ signaling in
fibroblasts is pro-tumorigenic in the mammary gland.
PKCβ IS INCREASED IN PyMT TUMOR-ASSOCIATED FIBROBLASTS AND
PATIENT BREAST CANCER STROMA
Given the decreased collagen deposition in the absence of stromal
PKCβ as just discussed, in addition to previously described roles
for PKCβ in fibroblast function (31–34), we further hypothesized
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PKCβ promotes mammary tumorigenesis
PyMT tumor volume isolated from wild type (WT) and Prkcb −/− mice
(n = 13 and 12, respectively, *P < 0.05 obtained from Mann–Whitney
test).
that MMTV–PyMT tumorigenesis may be, at least in part, due
to aberrant PKCβ expression in the mammary fibroblasts. To
this end, we isolated primary mammary tumor epithelial cells
and tumor-associated fibroblasts from PyMT mice 2 weeks after
palpable tumor formation. We also isolated mammary epithelial
cells and fibroblasts from age-matched WT littermate controls.
We assayed the expression level of Prkcb mRNA in both cellular compartments by quantitative real-time PCR (qRT-PCR), and
observed a significant decrease in Prkcb expression in mammary
epithelial cells derived from PyMT tumors (Figure 6A), possibly indicating that epithelial PKCβ is tumor suppressive during
PyMT -driven tumor formation. In contrast, a dramatic increase in
Prkcb mRNA in PyMT tumor-associated fibroblasts was observed
compared to WT mammary fibroblasts (Figure 6B). These data,
in combination with the decreased tumor formation we observed
in Prkcb −/− mice, suggest a pro-tumorigenic role for PKCβ in
mammary tumor fibroblasts. To confirm these findings in human
disease, we evaluated PRKCB in a publicly available breast cancer dataset [Karnoub et al. (43)] comparing stroma isolated from
invasive ductal breast carcinoma compared to normal breast tissue.
In this dataset, PRKCB is significantly increased in breast cancer
stroma versus normal confirming the translational relevance of
PKCβ in the breast TME (Figure 6C).
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PKCβ promotes mammary tumorigenesis
FIGURE 4 | Loss of PKCβ in stromal compartments has no effect on
tumor cell proliferation, vascularization, or macrophage infiltration.
(A) Left: representative images of Ki-67 staining of proliferating cells within
B6 PyMT tumors in vivo. Scale bar, 50 µm. Right: graph of percentage of
Ki-67+ cells relative to total DAPI+ cells (n = 3 per group, bars represent
means + standard deviation, P > 0.05 obtained from two-sample t -test).
(B) Left: representative images of F4/80 staining of macrophage cells within
B6 PyMT tumors in vivo. Scale bar, 50 µm. Right: graph of percentage of
F4/80+ area relative to total area of image (n = 3 per group, bars represent
means + standard deviation, P > 0.05 obtained from two-sample t -test).
(C) Left: representative images of MECA-32 staining of B6 PyMT tumor
vasculature in vivo. Scale bar, 50 µm. Right: graph of percentage of
MECA-32+ area relative to total area of image (n = 3 per group, bars represent
means + standard deviation, P > 0.05 obtained from two-sample t -test).
FIGURE 5 | Loss of PKCβ in stromal compartments decreases collagen
deposition. (A) Left: representative images of Masson’s Trichrome staining
within PyMT; Prkcb + / + and PyMT; Prkcb −/− tumors in vivo. Scale bar, 50 µm.
Right: graph of percentage of Trichrome collagen (blue) staining relative to
total area (n = 3 and 4, respectively, bars represent means + standard
deviation, P = 0.18 obtained from two-sample t -test). (B) Left: representative
images of Masson’s Trichrome staining within B6 PyMT tumors in vivo. Scale
bar, 50 µm. Right: graph of percentage of Trichrome collagen (blue) staining
relative to total area. n = 3 per group, bars represent means + standard
deviation, P < 0.05 obtained from two-sample t -test).
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FIGURE 6 | PKCβ is increased in PyMT tumor-associated fibroblasts and
patient breast cancer stroma. (A) Prkcb expression in primary mammary
fibroblasts normalized to Rpl4 and relative to WT. Cells isolated from PyMT
mice 2-weeks after first palpable tumor and WT littermate controls (n = 4,
bars represent means + standard deviation, *P < 0.05 obtained from
two-sample t -test). (B) Prkcb expression in primary mammary epithelial cells
DISCUSSION
The PKC family is well-documented to have a variety of roles in
cancer [reviewed in Ref. (1, 4)] with PKCβ functioning specifically in a number of cancer types including in breast (20–23,
45). Prior evaluation of PKCβ in breast cancer has utilized overexpression studies in vitro (20, 45) and expression analyses in
patient tumor tissue: both by immunohistochemistry of tumor
sections (21) and through whole tumor extraction (20, 23). While
the in vitro breast cancer cell line data are highly suggestive of a
tumor promoting role for PKCβ in breast cancer, tumor expression at both RNA (23) and protein (21) levels does not correlate
with breast cancer patient survival. Given that PKCβ is required for
VEGF-induced angiogenesis (25–27), immunity via NF-κB signaling (28–30), pro-tumorigenic fibroblast properties (31, 32), and
adipocyte lipid metabolism (35, 36), it is likely that our understanding of how PKCβ functions in breast tumorigenesis requires
further evaluation of its role in the breast TME.
In this study, we took advantage of the genomic Prkcb −/−
mouse (37). These mice are viable allowing for investigation of
mammary tumorigenesis in the absence of PKCβ either throughout the whole animal as we investigated in MMTV–PyMT mice
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PKCβ promotes mammary tumorigenesis
normalized to Rpl4 and relative to WT. Cells isolated from PyMT mice
2-weeks after first palpable tumor and WT littermate controls (n = 4, bars
represent means + standard deviation, *P < 0.05 obtained from two-sample
t -test). (C) Box and whisker plot of PRKCB expression in invasive ductal
breast carcinoma stroma compared to normal breast stroma in the Karnoub
breast dataset (normal, n = 15; IDC, n = 7, *P < 10−5 ).
with and without PKCβ, or through transplantation approaches
where we injected a MMTV–PyMT derived tumor cell line (B6
PyMT cells) into either WT or PKCβ null mammary fat pads. The
Prkcb −/− mice were crossed with the MMTV–PyMT mouse model
of breast cancer, which is a valuable translational tool given that
tumor formation in these mice recapitulates disease progression as
seen in humans (44). Furthermore, the importance of the mammary TME in MMTV–PyMT -induced tumorigenesis has been
well-described. Alterations in signaling from the endothelium
(46), fibroblasts (41, 47, 48), immune cells (49–52), and adipocytes
(53–55) have all been shown to alter tumor growth and/or metastatic progression in these mice. Moreover, increased stromal collagen deposition hastens MMTV–PyMT tumor development and
metastatic spread (56).
As described herein, MMTV–PyMT ; Prkcb −/− mice develop
smaller tumors at a longer latency than their MMTV–PyMT ;
Prkcb +/+ counterparts. This decrease in tumorigenesis is associated with a reduction in proliferation and macrophage recruitment. Tumor-associated macrophages in breast cancer act in a
pro-tumor manner by upregulation of M2 functions such as
promoting angiogenesis, remodeling of the tumor matrix, and
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suppressing the adaptive immune response (57, 58). Furthermore,
M2 macrophages are thought to have an immunomodulatory
function that activates tumor cell proliferation (59) suggesting a
possible mechanistic connection between the observed differences
in proliferation and macrophage number upon loss of PKCβ. Surprisingly, even with the well-described role for PKCβ in VEGF
signaling (25–27), there was no alteration in the tumor vasculature in PKCβ null PyMT mice. Similar findings were observed
in the injection model. However, in this case we saw no difference in proliferation, macrophage recruitment, or angiogenesis,
but did see a decrease in collagen deposition. These results imply
a mechanistic role for PKCβ signaling in fibroblast activity, specifically in the synthesis of collagen. It is important to note that
this model only evaluates tumor progression, not initiation, since
the B6 PyMT cells are already transformed. Future studies are
required using Prkcb floxed mice to conditionally delete PKCβ in
the endothelial (Tie2-cre), macrophage (Lys-cre), and fibroblast
(Fsp-cre) specific cellular compartments. These mice can then be
crossed with the MMTV–PyMT as well as other murine models
of breast cancer to investigate how PKCβ alters mammary tumor
initiation. While this investigation is well beyond the scope of
the current study, it will be necessary to truly define the specific
breast TME component where PKCβ has the most tumor altering
effect.
Support for a pro-tumorigenic role for PKCβ in the breast TME
is evidenced by the significantly upregulated expression of Prkcb
in fibroblasts isolated from MMTV–PyMT mammary tumors
when compared to WT mammary glands. Increased PRKCB in
breast tumor stroma versus normal stroma was confirmed in a
breast cancer patient cohort. Interestingly, we observed a significant decrease in Prkcb expression in the epithelial fraction of
the MMTV–PyMT tumors. The concomitant decrease of Prkcb in
epithelium and increase in the stroma suggests that whole tumor
analysis of PKCβ either at the mRNA or protein level could be
misinterpreted as showing no observable difference in expression.
Likewise, observable differences may be reflective of expression
changes in specific cellular populations confounding any predictive power of PKCβ. This is supported by the fact that similar
to published studies (21, 23), we observed no significant correlation of PRKCB with patient outcome in several publicly available
breast cancer datasets (data not shown). Given its role in the TME
as described herein, PKCβ expression can only be truly defined
through immunohistological evaluation of whole tumor sections
in order to delineate tumor versus stromal expression. Pursuing
whether PKCβ expression is predictive in breast cancer is critical
because a number of PKCβ selective inhibitors have shown efficacy in pre-clinical and clinical trials for a multitude of cancer
types (4, 60). To date, no studies have proven efficacious in breast
cancer patients, but this could be due to the absence of a proper
biomarker as tumor expression of PKCβ has not been evaluated
in trial participants [(61, 62); clinicaltrials.gov]. Continued investigation into the mechanism of PKCβ function in breast tumor
stroma through the use of the conditional knockout models as
described above will undoubtedly translate to a greater understanding of how to clinically evaluate PKCβ tumor expression and
ultimately use this aberrant signaling pathway as a therapeutic
target.
Frontiers in Oncology | Molecular and Cellular Oncology
PKCβ promotes mammary tumorigenesis
ACKNOWLEDGMENTS
We graciously thank Tsonwin Hai for the B6 PyMT cells as well
as the Solid Tumor Pathology Core at OSU for their technical
expertise. This work was supported by the National Institutes of
Health (PO1CA09719 to Michael C. Ostrowski) and an OSU CCC
Pelotonia Postdoctoral Fellowship (Gina M. Sizemore).
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Frontiers in Oncology | Molecular and Cellular Oncology
PKCβ promotes mammary tumorigenesis
Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 17 January 2014; paper pending published: 15 February 2014; accepted: 08
April 2014; published online: 23 April 2014.
Citation: Wallace JA, Pitarresi JR, Sharma N, Palettas M, Cuitiño MC, Sizemore ST,
Yu L, Sanderlin A, Rosol TJ, Mehta KD, Sizemore GM and Ostrowski MC (2014) Protein Kinase C beta in the tumor microenvironment promotes mammary tumorigenesis.
Front. Oncol. 4:87. doi: 10.3389/fonc.2014.00087
This article was submitted to Molecular and Cellular Oncology, a section of the journal
Frontiers in Oncology.
Copyright © 2014 Wallace, Pitarresi, Sharma, Palettas, Cuitiño, Sizemore, Yu, Sanderlin, Rosol, Mehta, Sizemore and Ostrowski. This is an open-access article distributed
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