Protein kinase C Beta in the tumor microenvironment promotes mammary tumorigenesis

Abstract

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: breast cancer; fibroblasts; mammary neoplasms (experimental); protein kinase C beta; stroma; tumor microenvironment.

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 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).

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. 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).

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 PyMT tumor volume isolated from wild type (WT) and Prkcb^−/− mice (n = 13 and 12, respectively, *P < 0.05 obtained from Mann–Whitney test).

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).

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 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).