Breast cancer-associated fibroblasts: their roles in tumor initiation, progression and clinical applications

Abstract

Breast cancer is the most common malignant tumor in women, and the incidence of this disease has increased in recent years because of changes in diet, living environment, gestational age, and other unknown factors. Previous studies focused on cancer cells, but an increasing number of recent studies have analyzed the contribution of cancer microenvironment to the initiation and progression of breast cancer. Cancer-associated fibroblasts (CAFs), the most abundant cells in tumor stroma, secrete various active biomolecules, including extracellular matrix components, growth factors, cytokines, proteases, and hormones. CAFs not only facilitate the initiation, growth, angiogenesis, invasion, and metastasis of cancer but also serve as biomarkers in the clinical diagnosis, therapy, and prognosis of breast cancer. In this article, we reviewed the literature and summarized the research findings on CAFs in breast cancer.

Keywords

cancer-associated fibroblast / breast cancer / progression / prognosis

Introduction

Breast cancer is the most common malignant tumor in women, and the incidence of this disease has increased in recent years worldwide because of changes in diet, living environment, old gestational ages, and other unknown factors. Research on the biological features of breast cancer has recently progressed remarkably, but some issues remain unresolved. Most previous studies focused on cancer cells [ 1], but an increasing number of recent studies have analyzed the contribution of tumor microenvironment to the initiation and progression of breast cancer [ 2– 4]. Tumor microenvironment consists of the extracellular matrix (ECM) and various tumor stromal cells, including fibroblasts, immune cells, inflammatory cells, endothelial cells, pericytes, adipocytes, and bone marrow-derived cells [ 5– 7]. Cancer-associated fibroblasts (CAFs), the most abundant cells in tumor stroma, secrete various ECM components, growth factors, cytokines, proteases, and hormones. CAFs not only promote the initiation, growth, angiogenesis, invasion, and metastasis of breast cancer but also serve as biomarkers in the clinical diagnosis, therapy, and prognosis of breast cancer [ 2– 4, 8]. Considering the importance of awareness on research developments in breast cancer, we present a literature review of the roles of CAFs in breast cancer.

Biological characters of CAFs in breast cancer

Biological features

Most fibroblasts in tumor stroma are activated, and only few are quiescent. CAFs are activated fibroblasts that produce various ECM components, such as collagen, proteoglycan, proteases, growth factors, and cytokines. CAFs share features of both smooth muscle cells and fibroblasts; therefore, they are also known as “myofibroblasts, peri-tumoral fibroblasts, reactive fibroblasts, or tumor-associated fibroblasts” [ 2]. Myofibroblasts were first discovered in granulation tissue during wound healing [ 9] and then confirmed to be activated fibroblasts. In addition to their existence in healing wound, myofibroblasts are also found in the stroma of malignant tumors [ 10]. The activation of CAFs in breast cancer is irreversible and results in prolonged life span through reduced apoptosis [ 4, 10]. Compared with normal fibroblasts (NFs), CAFs acquire higher capacities of proliferation and migration, and produce larger amounts of ECM [ 11].

Morphology and molecular markers

Under the microscope, CAFs appear as large spindle cells with abundant basophilic cytoplasm and indented nuclei. Electron microscopic evaluation indicates the presence of abundant rough endoplasmic reticulum, free ribosomes, well-developed Golgi complex, and rich tension fiber in the cytoplasm. Myofilaments and fibronexus junctions are observed in the peripheral cytoplasm [ 12, 13].

CAFs differ from NFs by loss of CD34 expression and gain of a-SMA expression [ 10]. CAFs also express other molecular markers, such as vimentin, fibroblast specific protein, fibroblast activation protein (FAP), osteonectin, and desmin. However, different molecular markers have been identified in different cancer subtypes [ 14, 15], and specific molecular markers that can recognize all of the CAFs in breast cancer remain lacking to date.

Cellular origins and activation mechanisms

Stromal CAFs of breast cancer have four distinct cellular origins [ 16]. The first one is activated normal stromal fibroblasts. Approximately 80% of fibroblasts in breast cancer stroma can be converted to this type, which is believed to be the primary source of CAFs [ 17– 20]. The second one is mammary epithelial cells that undergo epithelial-mesenchymal transition (EMT) or endothelial cells that undergo endothelial-mesenchymal transition [ 10, 21– 23]. The third one is bone marrow-derived mesenchymal stem cells (MSCs) [ 24– 27]. The last one includes transdifferentiated cells in breast tissue, such as pericytes, adipocytes, or smooth muscle cells [ 19].

NFs in breast stroma can be activated into CAFs through multiple pathways. In vitro or in vivo studies have indicated that transforming growth factor-b (TGF-b) and stromal cell-derived factor-1 (SDF-1/CXCL12), two cytokines secreted by fibroblasts, are important inducers of NF activation [ 18, 20]. Other cytokines produced by cancer cells, such as platelet-derived growth factor-a/b (PDGF-a/b), basic fibroblast growth factor (b-FGF), and interleukin-6 (IL-6), also activate NFs through paracrine effects [ 28– 32]. Another potential pathway is the activation of NFs through the downregulation or loss of expression of tumor suppressor genes, such as PTEN, caveolin-1 (Cav-1), p53, and p21, through various mechanisms [ 3, 33– 38]. Wang et al. [ 39] have recently reported that c-Ski-induced SDF-1 upregulation and p53 downregulation can convert NFs to CAFs. Other studies have suggested that the activation of NFs is associated with carcinogen exposure, local hypoxia, oxidative stress, aging, hormonal imbalance, and stromal cell DNA methylation [ 40, 41].

Association of CAFs with the development and progression of breast cancer

Tumor initiation

Mammary NFs can induce the reversion of the malignant phenotype in breast cancer [ 42], and CAFs promote the proliferation and malignant transformation of mammary epithelial cells. Kuperwasser et al. [ 43] found that sublethal doses of irradiation induce NFs to overexpress TGF-b and hepatocyte growth factor (HGF); in addition, normal mammary epithelial cells co-transplanted with activated NFs into animals undergo malignant transformation. By contrast, no tumor formation occurs when the same epithelial cells are co-transplanted with NFs. Nguyen et al. [ 44] found using a co-transplanting mouse model that irradiated fibroblasts have a significantly higher tumor inducing rate than unirradiated NFs. Meanwhile, Tyan et al. [ 45] revealed using co-culture and co-implantation models that MDA-MB-468 breast cancer cells induce CAFs to secrete HGF and enhance tumorigenecity. These studies indicate that the conversion of NFs into CAFs may occur at the initiation phase of breast cancer preceding the genetic alterations of epithelial cells and that CAFs may induce the malignant transformation of adjacent mammary epithelial cells. Furthermore, the occurrence of breast cancer is closely related to estrogen stimulation. Shekhar et al. [ 46] demonstrated in a 3D cell-cell interaction model that NFs inhibit estrogen-induced tumor cell growth while CAFs synthetize abundant estrogen and induce the malignant transformation of the normal mammary epithelial cell line MCF10A and the pre-cancerous mammary epithelial cell line EIII8.

In summary, these in vitro and in vivo studies suggest that CAFs play crucial roles in inducing the initiation of breast cancer.

Tumor growth

Uncontrolled growth is the precondition for malignant tumor invasion and metastasis. Many in vitro and in vivo investigations have shown that CAFs can promote breast cancer tumor growth. Orimo et al. [ 47] demonstrated using a co-implantation tumor xenograft model that CAFs extracted from human breast cancer induce significantly more tumor growth than NFs derived from the same patients. They concluded that CAFs not only induce mammary carcinogenesis but also promote breast cancer tumor growth. They further identified that CAFs promote tumor growth primarily through SDF-1 secretion, which can preferentially bind to CXCR-4 receptors in cancer cells and then stimulate their proliferation [ 47, 48]. In addition, SDF-1 induces tumor neovascularization and facilitates tumor growth indirectly [ 47]. Huang et al. [ 49] found that SDF-1 secreted by CAFs stimulates the proliferation of CD44⁺CD24^‒ breast cancer cells and prominently enhances the tumorigenicity in mice by co-injection of CAFs with mammosphere cells.

CAF-derived growth factors, such as HGF, FGF, and TGF, promote the proliferation of breast cancer cells. Stuelten et al. [ 50] found that in vivo breast cancer MCF10CA1a cells induce NFs to secrete TGF-b and accelerate tumor growth. CAF-produced FGF-1 and FGF7 stimulate profound breast cancer cell proliferation via paracrine activation [ 51, 52]. Tyan et al. [ 53] demonstrated using co-culture and co-implantation models that CAF-secreted HGF promotes the proliferation of cancer cells through HGF/Met signaling pathways. Breast cancer cells consequently induce further secretions of these growth factors by CAFs. These findings indicate that a reciprocal feedback loop exists between cancer cells and CAFs, which collectively promote tumor growth.

Tumor angiogenesis

Angiogenesis is essential and crucial for the growth, invasion, and metastasis of tumors. A tumor could not grow exceeding 1‒2 mm in size until angiogenesis promptly establishes in the tumor. In general, angiogenesis is induced by cancer cells [ 1, 54]. However, recent studies have suggested that stromal CAFs aside from cancer cells also participate in tumor angiogenesis. Orimo et al. [ 47] found using a co-implantation tumor xenograft model that CAFs significantly enhance tumor vascularization and recruit endothelial progenitor cells into the tumor. They associated this phenomenon with the SDF-1/CXCR4 signaling pathway; that is, CAF-secreted SDF-1 could bind to the vascular endothelial cell cognate receptor CXCR4 to promote angiogenesis and recruit endothelial progenitor cells. This pathway is also reportedly involved in the angiogenesis of prostate cancer. Wang et al. [ 55] demonstrated that knock-down of CXCR4 in cancer cells decreases the number of blood vessels formed after the cells are co-transplanted with vascular endothelial cells into mice. Their study suggested that the SDF-1/CXCR4 signaling pathway is directly involved in angiogenesis. Maeda et al. [ 56] used co-culture, tumor xenografts, and human breast carcinoma tissue array and found that the expression of syndecan-1 (Sdc-1) is highly upregulated in CAFs and is significantly associated with the proliferation of microvessels featuring increased vascular density and large area occupation. The authors concluded that CAF-derived Sdc-1 stimulates the tumor growth and angiogenesis of breast cancer.

Other CAF-derived growth factors, such as FGF, PDGF, TGF, and vascular endothelial growth factor, also play important roles in promoting the proliferation and differentiation of endothelial cells and in inducing the angiogenesis of breast tumors [ 10, 57].

Tumor invasion and metastasis

Invasion and metastasis, characters of malignant tumors, lead to tumor progression and patient death. Invasion and metastasis of cancer cells are extremely complicated processes that involve cancer cell separation, ECM degradation, intravasation into blood or lymphatic vessels, and metastatic tumor formation in distant organs. Breast CAFs participate in multiple steps of breast cancer invasion and metastasis.

First, cancer cells separate from other cells in the group through EMT and subsequent migration, which prepares for invasion into ECM [ 58, 59]. CAFs induce the EMT of cancer cells [ 59– 62]. Soon et al. [ 61] studied the association of molecular markers with the invasion of breast cancer MCF7 cells co-cultured with CAFs or NFs and found that CAFs induce significantly greater EMT of MCF7 cells than NFs. In addition, CAFs induce EMT by activating growth factors, such as TGF-b1, EGF, PDGF, HGF, and matrix metalloproteinases (MMPs) [ 59, 60, 63– 65]. Gao et al. [ 62] identified that CAFs located in human breast cancer interface zones between tumor and normal tissue are strong inducers of EMT.

Second, CAFs produce various MMPs that are involved in modulating tumor microenvironment [ 66]. MMPs can directly depredate the ECM to suit cancer cell migration and destroy the basement membrane of blood or lymphatic vessels for cancer cell intravasation [ 67]. In vitro and in vivo studies have indicated that CAFs promote breast cancer cell invasion and metastasis by MMPs, such as MMP-1, -2, -3, -7, -9, -13, and-14 [ 66– 70]. For instance, Wang et al. [ 70] found that CAF-derived MMP-9 facilitates breast cancer cell invasion in a culture of human breast cancer MDA-MB-231 cells alone and in a co-culture of these cells with human fibroblasts. Similarly, Eck et al. [ 69] reported that CAF-derived MMP-1 promotes breast cancer cell invasion and migration.

Third, CAFs form clusters with cancer cells during tumor invasion into blood or lymphatic vessels. CAFs co-travel with cancer cells and enhance vessel invasion by protecting cancer cells from immune attack, enduring fluid mechanical force, and reducing cancer cell apoptosis. Duda et al. [ 71] demonstrated using a tumor metastasis mouse model that metastatic lung cancer cells preferentially grow if they co-metastasize with their own stromal CAFs from the primary site. Conversely, the number of metastatic cancer cells significantly decreases when the CAFs accompanying metastatic cancer cells are removed.

Finally, CAFs extravasate blood or lymphatic vessels along with cancer cells to form metastatic tumors in appropriate organs. CAFs survive in the new environment and continuously secrete growth factors and cytokines to promote the proliferation of metastatic cancer cells.

Paracrine activation is the primary mode of CAFs in promoting cancer cell invasion and metastasis by secreting growth factors, cytokines, and proteinases [ 50, 59, 60, 66]. However, recent studies have indicated that the mechanisms also involve mechanical pressure in cancer tissue [ 72, 73]. Luga et al. [ 74] have recently reported that CAFs secrete exosomes to promote the motility and metastasis of breast cancer cells by activating Wnt-planar cell polarity signaling.

Values of CAFs in the diagnosis, treatment, and prognosis of breast cancer

Clinical diagnosis and prognosis

The values of CAFs in the clinical diagnosis and prognosis of breast cancer have been studied extensively, and the potential usage of some CAF molecular markers has been identified. For example, breast CAFs express a-SMA, whereas NFs in breast tissue lack this expression. Yamashita et al. [ 75] identified through immunohistochemisty that a-SMA expression in myofibroblasts is an independent predictor of metastasis and poor prognosis in invasive breast cancer patients. In addition, CAF-derived PDGF receptor ß and FAP are significantly associated with breast cancer recurrence, disease-free survival, and overall survival [ 76– 79].

Mutation and/or loss of tumor suppressor genes are also important features of breast CAFs. Hasebe et al. [ 80] reported that p53 gene expression in CAFs is significantly correlated with tumor lymph node metastasis and poor prognosis in invasive ductal carcinoma patients. Shan et al. [ 81, 82] revealed that the expression of the tumor suppressor gene Cav-1 in breast CAFs is closely related to histological type, histological grade, ER status, and molecular subtype. The downregulated expression or absence expression of Cav-1 in CAFs indicates early disease recurrence, lymph node metastasis, and poor prognosis in breast cancer patients [ 81, 83, 84]. Witkiewicz et al. [ 85] stratified breast cancer patients into high-risk and low-risk groups on the basis of Cav-1 expression, with the high-risk patients lacking Cav-1 expression in CAFs, and suggested that extensive therapies are necessary to this group of patients. In a separate study, they found that the loss of Cav-1 expression in CAFs is an important prognostic factor for triple-negative and basal-like breast cancers [ 84]. Our group has recently demonstrated that the absence of Cav-1 expression in CAFs is associated with the large tumor size and lymph node metastasis of breast invasive micropapillary carcinoma; in addition, this condition may serve as an independent predictor of reduced progression-free survival [ 86].

Some types of CAF-derived MMPs have a significant value in the diagnosis and prognosis of breast cancer [ 8]. Ranogajec et al. [ 87] showed that the expression of MMP-2 or co-expression of MMP-2/MMP-9 in CAFs is significantly associated with tumor size. Bostrom et al. [ 88] analyzed 125 breast cancer specimens via immunohistochemistry and found that tumoral and stromal MMP-1 expression is associated with the tumor progression and poor prognosis of patients. Zhang et al. [ 89] discovered through immunohistochemistry that MMP-13 expression in CAFs is associated with tumor lymph node metastasis. Scherz-Shouva et al. [ 90]have recently reported that the frequent activation of the transcriptional regulator heat shock factor 1 in CAFs is significantly associated with the poor outcome of breast cancer patients.

In summary, CAFs have the potential to be utilized in the clinical diagnosis and prognosis of breast cancer. Evaluating their molecular markers and derived bio-products may provide information for the individualized treatment of breast cancer patients.

Therapy

CAFs play important roles in the initiation, growth, angiogenesis, invasion, and metastasis of breast cancer. Compared with cancer cells, CAFs feature genetic stability and less drug resistance; therefore, CAFs may serve as an effective therapeutic target in breast cancer [ 91]. Therapy options may theoretically include blocking CAF formation in breast cancer stroma. Tanaka et al. [ 92] have found that IFN-g can effectively block the generation of myofibroblasts by inhibiting a-SMA expression. Inhibition of the TGF-b/Smad pathway through bone morphogenetic protein and activin membrane-bound inhibitor transduction interrupts the formation of CAFs from MSCs cells, which might be helpful to MSC-based therapies in breast cancer patients [ 93]. Meanwhile, c-Ski induces NF activation and thus might be another potential therapeutic target for breast cancer [ 39]. Tchou et al. [ 91] suggested that FAP depletion in CAFs results in stunted tumor growth. Reisfeld [ 94] found in mouse tumor cells that using DNA vaccines directly against these specific genes in CAFs inhibits tumor progression.

CAF-derived ECM components, enzymes, cytokines, and growth factors contribute to the progression of breast cancer. Therefore, these products could become potential therapeutic targets. For instance, SDF-1/CXCL12 is an important factor in the growth, angiogenesis, invasion and metastasis of breast cancer; therefore, preventing SDF-1/CXCL12 from binding to CXCR4 could eliminate its tumor promoting effect. Burger et al. [ 95] identified using a preclinical tumor model the anti-tumor effects of CXCR4 antagonists, which have become effective therapeutic agents in various malignancies.

Therapeutic resistance is a main cause of poor therapeutic results or even failure in breast cancer patients. CAFs are involved in the resistance to endocrine therapy, chemotherapy, and target therapy by different mechanisms [ 96– 100]; hence, induction of CAF apoptosis or target therapy directly against CAFs may become an effective therapeutic approach in breast cancer.

Conclusions

CAFs, the most abundant cells in breast cancer stroma, derive various ECM components, growth factors, cytokines, proteins, enzymes, and hormones. CAFs participate in the development and progression of breast cancer by stimulating epithelial cell malignant transformation, tumor initiation, tumor growth, ECM degradation, tumor angiogenesis, and cancer cell invasion and metastasis. Furthermore, CAFs are valuable in the clinical diagnosis, therapy, and prognosis of breast cancer. However, many issues remain unclear, such as the relationship between CAFs and other mesenchymal cells, the precise mechanism of their escape from immune attack, and whether or not other valuable molecular markers of CAFs exist. Thus, breast CAFs warrant further investigations.

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