Breast Cancer Stroma Research Articles

Tenascin-C (TNC) Promotes Breast Cancer Cell Invasion and Proliferation: Functional Effects of TNC Knockdown in Highly Invasive Breast Cancer Cell Lines

The tumour microenvironment plays a crucial role in the development of breast cancer. Tenascin-C (TNC), a matricellular protein and its high molecular weight (MW) isoforms have been shown to be over-expressed in the stroma of breast cancers and are associated with poor prognosis. The aim of this study was to investigate the effects of TNC knockdown in TNC expressing invasive breast cancer cell lines on cancer cell behaviour. Small interfering RNA (siRNA) targeting different exons in TNC (24, 14 and 14-AD1) were designed, synthesised and transfected into the highly invasive MDA-MB-231 breast cancer cell line. The phenotypic alterations caused by TNC knockdown were analysed by Two Dimension (2D) invasion assays and proliferation assays using the mitotic marker Phispho-Histone H3 (pHH3). The siRNA targeted cells showed significant down-regulation of both total TNC (p <0.001) and high MW isoforms (p <0.001) in MDA-MB-231 cells. Moreover, knockdown of total TNC and high MW TNC isoforms significantly decreased both cell invasion (total TNC p<0.001 and TNC-14 p <0.001) and proliferation (total TNC p <0.001 and TNC-AD1 p <0.05). In conclusion, TNC knockdown significantly decreases proliferation and invasion in breast cancer cell lines, confirming its importance in breast cancer progression.

Targeting the Tumor Stroma in Breast Cancer

Tumor stroma is constituted by blood vessels, fibroblasts, leukocytes, and intercellular fibers. As opposed to the stroma of normal tissue, tumor stroma has an aberrant physiology. The tumor cells can secrete growth factors that can reprogram the stroma so that it exerts several tumor progression roles and supports a positive feedback loop that preserve a microenvironment favorable for the tumor and hostile for the patient’s interests (limiting the delivery of chemotherapy, blocking immune response, and keeping hypoxic conditions). In this review, we discuss the recent advances in understanding this tumor-stromal cross-talk and how the signaling systems implicated on it can be useful for targeted therapeutics. We will discuss the available results from trials completed with targeted agents with antistromal effects and also retrospective studies conducted with patient cohorts taking other non-cancer drugs to which recently several antistromal properties have been described, such as antihypertensives. Finally, we will show our preclinical results regarding hypoxia tracking with 18F-fluoromisonidazole, a PET tracer, as a potential tool to specifically monitor the positive or negative effects of antistromal agents.

Abstract A62: TGF-β regulates CXCL1 expression in mammary carcinoma associated fibroblasts through novel Smad2/3- and HGF/c-Met-dependent mechanisms.

Abstract CXCL1 is a chemokine secreted by macrophages, neutrophils, epithelial cells and fibroblasts, and plays a role in inflammation and wound healing. Enhanced expression of CXCL1 in tumor epithelium is associated with cell invasion, angiogenesis in melanoma, bladder, ovarian and breast cancer. However, little is known about CXCL1 expression in tumor stroma. Through analysis of gene expression data, our studies reveal that high RNA levels of CXCL1 in breast tumor stroma is associated with increased rate of recurrence and shorter relapse-free survival. We noticed decreased secretion levels of TGF-β and increased secretion of CXCL1 in tumor derived fibroblasts. Increased CXCL1 expression was also observed in mammary fibroblasts in which the TGF-β type II receptor was knocked out. By immunohistochemistry analysis we found that expression of CXCL1 inversely correlated with expression of TGF-β, phosphorylated-Smad2 and phosphorylated-Smad3 in breast tumor stroma. We hypothesized that TGF-β suppresses CXCL1 expression in breast fibroblasts. Our goal for this project was to determine the molecular mechanisms through which TGF-β suppresses CXCL1 expression using mammary fibroblasts derived from MMTV-PyVmT mammary tumor model. Treatment of fibroblasts with recombinant TGF-β decreased CXCL1 transcription and secretion by up to 85% over time, as determined by RT-PCR and ELISA. SiRNA silencing of TGF-β signaling transducers, Smad2 and Smad3, increased CXCL1 transcription and secretion in mammary fibroblasts. In addition, TGF-β suppressed expression of HGF, a factor that enhanced CXCL1 expression in mammary fibroblasts. Knockdown of HGF or treatment of fibroblasts with c-Met inhibitors reduced CXCL1 expression. These data indicate TGF-β suppresses CXCL1 expression through Smad2/3-dependent mechanisms. As a secondary mechanism, TGF-β suppresses CXCL1 expression through down-regulation of HGF/c-Met signaling. This is the first report to characterize the mechanisms of CXCL1 expression in mammary fibroblasts and demonstrate a clinical relevance for CXCL1 expression in breast cancer stroma. Citation Format: An Zou, Diana Lambert, Henry Yeh, Wei Bin Fang, Nikki Cheng. TGF-β regulates CXCL1 expression in mammary carcinoma associated fibroblasts through novel Smad2/3- and HGF/c-Met-dependent mechanisms.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A62. doi:10.1158/1538-7445.CHTME14-A62

Tumor-stroma crosstalk: targeting stroma in breast cancer.

Combinatorial strategies in cancer medicine will not only target cancer cell-intrinsic pathways, but also cancer cell-extrinsic cells, pathways, and mediators of the tumor microenvironment. The aim of the present review is to define the roles of the tumor microenvironment in primary and metastatic breast cancer progression. The cancer microenvironment is composed of nontransformed host stromal cells, such as endothelial cells, fibroblasts, various immune cells, and a complex extracellular matrix secreted by both the normal and neoplastic cells embedded in it. The stromal constituents contribute to the core and emergent hallmarks of cancer. In particular, they can boost sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming energy metabolism, and evading immune destruction. The stromal cells play a role in enabling or enhancing multiple hallmark capabilities in tumor microenvironment. This is a background for therapeutic-targeting strategies aimed to abrogate the stroma's contribution. Targeting tumor-associated fibroblasts, macrophages, angiogenesis and enhancing immune response may represent a paradigm-shifting approach to treating human cancer in the near future.

Elevated CXCL1 expression in breast cancer stroma predicts poor prognosis and is inversely associated with expression of TGF-β signaling proteins.

BackgroundCXCL1 is a chemotactic cytokine shown to regulate breast cancer progression and chemo-resistance. However, the prognostic significance of CXCL1 expression in breast cancer has not been fully characterized. Fibroblasts are important cellular components of the breast tumor microenvironment, and recent studies indicate that this cell type is a potential source of CXCL1 expression in breast tumors. The goal of this study was to further characterize the expression patterns of CXCL1 in breast cancer stroma, determine the prognostic significance of stromal CXCL1 expression, and identify factors affecting stromal CXCL1 expression.MethodsStromal CXCL1 protein expression was analyzed in 54 normal and 83 breast carcinomas by immunohistochemistry staining. RNA expression of CXCL1 in breast cancer stroma was analyzed through data mining in http://www.Oncomine.org. The relationships between CXCL1 expression and prognostic factors were analyzed by univariate analysis. Co-immunofluorescence staining for CXCL1, α-Smooth Muscle Actin (α-SMA) and Fibroblast Specific Protein 1 (FSP1) expression was performed to analyze expression of CXCL1 in fibroblasts. By candidate profiling, the TGF-β signaling pathway was identified as a regulator of CXCL1 expression in fibroblasts. Expression of TGF-β and SMAD gene products were analyzed by immunohistochemistry and data mining analysis. The relationships between stromal CXCL1 and TGF-β signaling components were analyzed by univariate analysis. Carcinoma associated fibroblasts isolated from MMTV-PyVmT mammary tumors were treated with recombinant TGF-β and analyzed for CXCL1 promoter activity by luciferase assay, and protein secretion by ELISA.ResultsElevated CXCL1 expression in breast cancer stroma correlated with tumor grade, disease recurrence and decreased patient survival. By co-immunofluorescence staining, CXCL1 expression overlapped with expression of α-SMA and FSP1 proteins. Expression of stromal CXCL1 protein expression inversely correlated with expression of TGF-β signaling components. Treatment of fibroblasts with TGF-β suppressed CXCL1 secretion and promoter activity.ConclusionsIncreased CXCL1 expression in breast cancer stroma correlates with poor patient prognosis. Furthermore, CXCL1 expression is localized to α-SMA and FSP1 positive fibroblasts, and is negatively regulated by TGF-β signaling. These studies indicate that decreased TGF-β signaling in carcinoma associated fibroblasts enhances CXCL1 expression in fibroblasts, which could contribute to breast cancer progression.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-781) contains supplementary material, which is available to authorized users.

Abstract 1067: Somatic genomic mutations, RNA-Seq, and proteomic signal transduction network analysis of tumor and stroma in metastatic triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous, basal like tumor category for which there is no molecular targeted therapy that has significantly improved overall patient survival. While genomic sequencing has been conducted on primary triple negative breast cancers, these data may not correspond to metastatic TNBC because the metastatic colonies may comprise a subclone of the primary. Moreover, it has been difficult to identify the true driver mutations in the primary tumor. We have hypothesized that evaluation of the protein signal transduction pathways within the tumor-stromal microenvironment of the metastatic tumor may be vastly different than the primary tumor and can provide potential clues to the driver mutations and stromal interactions. Somatic genomic mutations and RNA-Seq profiles were previously cataloged for a set of 14 fresh frozen metastatic TNBC samples. TP53, LRP1B, HERC1, CDH5, RB1, and NF1 were mutated in multiple tumors. FOXM1 gene overexpression was noted in tumor samples compared to non-malignant breast samples. To complement the genetic data, we evaluated more than 70 signal transduction proteins in tumor and stroma for 13 of the metastatic TNBC samples from this cohort. The samples represented lymph node (n=5) and local and distant soft tissue (n=8) metastasis. For proteomic analysis, purified populations of tumor and adjacent stromal cells were collected by Laser Capture Microdissection. Cell signaling pathway proteins were quantified by reverse phase protein microarray. Proteins from diverse pathways were included to: a) correlate our proteomic data with the previous genomic data, b) potentially identify previously unrecognized pathways involved in TNBC metastatic disease, and c) evaluate the tumor-stroma microenvironment in metastatic TNBC. We compared the signaling network of the tumor and its matched stroma to investigate tumor-stroma crosstalk. We also compared the signal pathways to the genomic abnormalities of the matched lesion. We confirmed the down regulation of endocrine (ER, AR, ProgRec) and Her2 pathways. phosphoJAK2 and Her4 (total) were both markedly downregulated. Signal pathway data provided information about the functional importance of the genomic data. Understanding the molecular underpinnings of metastatic TNBC may help elucidate genomic and proteomic alterations which can potentially lead to new strategies for individualizing therapy of metastasis. Citation Format: Virginia A. Espina, Joyce A. O'Shaughnessy, Maren K. Levin, David W. Craig, John D. Carpten, Daniel Von Hoff, Lance A. Liotta. Somatic genomic mutations, RNA-Seq, and proteomic signal transduction network analysis of tumor and stroma in metastatic triple negative breast cancer. [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 1067. doi:10.1158/1538-7445.AM2014-1067

Abstract 181: Therapeutic approaches to metastasis induced by mesenchymal stem cells in the tumor microenvironment

Abstract Metastasis is the primary cause of death in non-hematological cancers yet there are no specific therapeutics against it because of a lack of validated targets. The Weinberg lab demonstrated that bone-marrow derived mesenchymal stem cells (MSCs) home to the stroma of breast tumors and induce metastasis. We verified these findings in MDA-MB-231 (MDA) xenografts in which co-injection of human MSCs increased MDA thoracic metastasis by 5-fold. In vitro, MSC co-culture induced GFP-labeled MDA cells to migrate 3-fold faster in a modified ‘wound-healing’ assay, mirroring metastasis in vivo. To identify therapeutic targets within MSC-induced metastasis, we performed gene-expression analysis of MSC-MDA co-cultures separated by flow cytometry compared with cells grown alone. The interferon pathway was found to be the most activated pathway upon co-culture, increasing mainly in MSCs. We studied the relevance of these genes in human cancers by first analyzing 3 different gene-expression datasets comparing human breast cancer stroma with normal stroma. Genes upregulated in breast cancer stroma were then studied in a meta-analysis of 19 whole tumor gene-expression datasets correlating gene-expression changes with survival. We identified 103 genes that are upregulated in MSCs by MSC-MDA interactions, are increased in human breast cancer stroma and are significantly associated with poor survival. To determine if they are necessary for MSC-induced metastatic behavior, we performed shRNA knockdown in MSC-MDA co-cultures measuring effects on in vitro migration. Knockdown of a number of interferon-associated genes significantly reduced migration supporting an unexpected functional role of interferons in metastasis. The top interferon gene, ISG15, is an attractive candidate for therapeutic targeting because it is a secreted ubiquitin-like factor that conjugates a number of cytoskeletal proteins involved in motility. In parallel, we conducted a small-molecule screen with 1600 compounds on the migration of MSC-MDA co-cultures to identify small-molecule inhibitors of metastasis. Counter screens on highly motile endothelial cells excluded compounds that non-specifically inhibit normal cell migration. Only 1 compound, RSL3, specifically blocked MSC-induced MDA migration with cytotoxicity at &amp;gt;10-fold higher concentrations. RSL3 inhibits the glutathione peroxidase 4 (GPX4) enzyme that metabolizes lipid peroxides including arachidonic acid metabolites participating in inflammatory cascades like interferon gamma signaling. RSL3 activity was completely abrogated by co-treatment with the 5-lipoxygenase inhibitor zileuton consistent with the role of arachidonic acid metabolites in MSC-MDA migration. In summary, targeting components of interferon and arachidonic acid pathways have been discovered as novel therapeutic approaches against microenvironment-induced breast cancer metastasis. Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Siddhartha Mukherjee, Rushdia Z. Yusuf, Vasanthi Viswanathan, Ben S. Wittner, Jeff Gentry, Alykhan Shamji, Sridhar Ramaswamy, David T. Scadden, Stuart L. Schreiber. Therapeutic approaches to metastasis induced by mesenchymal stem cells in the tumor microenvironment. [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 181. doi:10.1158/1538-7445.AM2014-181

Clinicalpathological Characteristics of Cancer-Associated-Fibroblast in Esophageal Squamous Cell Carcinoma

ABSTRACT Aim: Cancer associated fibroblasts (CAFs) are a subpopulation of cells that reside within the tumor microenvironment and promotes the transformation process by encouraging tumor growth, neoangiogenesis, modulation of cancer stem cell traits, and formation of metastases. Although CAFs have been observed within the stroma of breast, lung, and pancreas cancers, the precise clinicalpathological characteristics of CAFs in esophageal squamous cell carcinoma (ESCC) remains unclear, and its source is also require further elucidation. Methods: Expression of CAFs markers, a-SMA and S100A4, were examined in 68 ESCC specimens by immunocytochemistry, and the association of a-SMA and S100A4 expression with clinical and pathological features and prognosis were also evaluated. Cell cultured supernate of ESCC cell lines, including Eca-109?TE-1, were collected and then cultured with mesenchymal stem cells (MSCs). Temporal correlation morphological changing of MSCs was observed and expression of a-SMA and S100A4 were also examined by quantitative PCR and Western blot. Results: High a-SMA and S100A4 levels in tumor stroma were expressed in 47.0 % and 42.6 % ESCC samples (32/68 cases, 29/68 cases, respectively), and their expression distribution in each sample was consistent. a-SMA expression correlated significantly with a poorly differentiated phenotype (k2=5.13, P=0.023) and high TNM classification (k2=2.66 P=0.049). Kaplan-Meier plots revealed large differences in OS estimates between patients with high and low a-SMA expression (P=0.029). More importantly, quantitative PCR showed increasing of a-SMA and S100A4 mRNA levels in MSCs after culturing and inducing with cell supernate of Eca-109 and TE-1 cell lines, and Western blot also implied enlargement of a-SMA and S100A4 levels in MSCs after culturing and inducing. Conclusions: There is prominent distribution of CAFs in ESCC tissues, and these CAFs would make a contribution on poor prognosis of patients. Tumor cell induction data demonstrates that mesenchymal stem cells may be the candidate of CAFs in ESCC tissues. The precise mechanisms of CAFs in tumor tissues action in the invasion and metastasis of ESCC require further elucidation. Disclosure: All authors have declared no conflicts of interest.

Alterations in collagen fibre patterns in breast cancer. A premise for tumour invasiveness?

Stromal tissue in the breast plays a key role in cancer invasiveness due to molecular and cellular changes. Collagen is the main component of the stroma. The purposes of this study were to investigate differences in collagen fibre patterns between tumour-induced stromal tissue and normal stroma, and between high-grade and low-grade breast cancer stroma, using second harmonic generation microscopy. Thirty-seven ductal carcinomas were examined: Twenty-one Luminal A phenotype and sixteen HER2 or Basal-like phenotype. Three regions were examined in each case: intratumoral, juxtatumoral and extratumoral. Two images were captured in each region. Two characteristics of collagen fibres were examined: the degree of straightness, and the degree of alignment. Collagen fibres were visually classified as curly, intermediate or straight, and as parallel or not parallel. The results of angle measurement and visual analysis showed that collagen fibres were straightest in the intratumoral region and curliest in the extratumoral region. Collagen fibres were more parallel in the juxtatumoral region compared to the two other regions. There were no significant differences between high-grade and low-grade tumours. As a breast tumour progresses, collagen fibres appear to straighten and align at the tumour boundary. This could facilitate invasion of the tumour into the surrounding stroma.

Cancer-associated fibroblasts from invasive breast cancer have an attenuated capacity to secrete collagens.

Normal fibroblasts produce extracellular matrix(ECM) components that form the structural framework of tissues. Cancer-associated fibroblasts(CAFs) with an activated phenotype mainly contribute to ECM deposition and construction of cancer masses. However, the stroma of breast cancer tissues has been shown to be more complicated, and the mechanisms through which CAFs influence ECM deposition remain elusive. In this study, we found that the activated fibroblast marker α-smooth muscle actin(α-SMA) was only present in the stroma of breast cancer tissue, and the CAFs isolated from invasive breast cancer sample remained to be activated and proliferative in passages. To further assess the difference between CAFs and normal breast fibroblasts(NFs), MALDITOF/TOF‑MS was used to analyze the secretory proteins of primary CAFs and NFs. In total, 2,903 and 3,023 proteins were identified. Mass spectrum quantitative assay and data analysis for extracellular proteins indicated that the CAFs produce less collagens and matrix-degrading enzymes compared with NFs. This finding was confirmed by western blot analysis. Furthermore, we discovered that reduced collagen deposition was present in the stroma of invasive breast cancer. These studies showed that although CAFs from invasive breast cancer possess an activated phenotype, they secreted less collagen and induced less ECM deposition in cancer stroma. In cancer tissue, the remodeling of stromal structure and tumor microenvironment might, therefore, be attributed to the biological changes in CAFs including their protein expression profile.

Functional comparison between genomic subtypes and protein signal transduction networks in metastatic triple-negative breast cancer tumor and stroma to identify actionable therapeutic strategies.

e22155 Background: Genomic sequencing has been conducted on primary TNBC, however it has been difficult to identify driver mutations that constitute actionable therapy strategies. Importantly, geno...

CYP3A4 expression in breast cancer and its association with risk factors in Mexican women.

In Mexico, breast cancer (BCa) is the leading type of cancer in women. Cytochrome P450 (CYP450) is a superfamily of major oxidative enzymes that metabolize carcinogens and many antineoplastic drugs. In addition, these enzymes have influence on tumor development and tumor response to therapy. In this report, we analyzed the protein expression in patients with BCa and in healthy women. Links with some clinic-pathological characteristic were also assessed. Immunohistochemical analyses were conducted on 48 sets of human breast tumors and normal breast tissues enrolled in Hospital Militar de Especialidades de la Mujer y Neonatologia and Hospital Central Militar, respectively, during the time period from 2010 to 2011. Informed consent was obtained from all participants. Statistical analysis was performed using χ2 or Fisher exact tests to estimate associations and the Mann Whitney U test for comparison of group means. We found a significant CYP3A4 overexpression in BCa stroma and gland regions in comparison with healthy tissue. A significant association between protein expression with smoking, alcoholism and hormonal contraceptives use was also observed. Additionally, we observed estrogen receptor (ER) and progesterone receptor (PR) positive association in BCa. We suggest that CYP3A4 expression promotes BCa development and can be used in the prediction of tumor response to different treatments. One therapeutic approach may thus be to block CYP3A4 function.

P26. Cancer associated fibroblasts contribute to the immune suppression of breast cancer by augmentation of the inflammatory products

Meeting abstracts The supporting stroma of breast cancer is believed to support the growth and metastasis of cancer cells and are responsible for suppressing anti-cancer immune responses. In this regard we attempted to isolate and characterise the cancer associated fibroblasts (CAFs) of murine

Steroid hormone receptor expression in breast cancer stroma

Around two-thirds of all breast carcinomas respond to adjuvant endocrine therapy with the presence of ERa in tumour epithelial cells used to guide therapy. Other members of the steroid receptor superfamily have also been detected in breast cancer epithelial cells, notably PR, AR, whose presence and potential prognostic role in breast cancer is gaining momentum [1, 2], and there is ongoing interest in the potential role of ERb in breast carcinogenesis [3]. However, scientists are now looking beyond the epithelial landscape with the recognition that interaction of tumour epithelial cells with its surrounding microenvironment, particularly the stroma plays a role in promoting tumour growth and progression. While recognising that the stroma is multicellular, much of this attention has focused on fibroblasts residing in tumour stroma, termed carcinoma-associated fibroblasts (CAFs) as there is growing appreciation that CAFs may provide a favourable environment to augment tumour growth [4]. A recent report in Breast Cancer Research and Treatment by Knower et al. [5] used low density Taqman arrays followed by qPCR and immunohistochemical validation to examine the presence of a panel of 48 nuclear receptors in CAFs, derived from ERa-positive breast carcinomas and in normal breast adipose fibroblasts (NAFs). Receptor expression was grouped as absent, low, moderate or high. Intriguingly, of the four nuclear hormone receptors previously associated with breast carcinogenesis listed above, none were identified as being highly expressed in breast stroma; ERb was absent, ERa and PR showed low expression, while moderate expression of AR was seen. As a group with a major interest in the role of ERs in breast carcinogenesis, we have routinely monitored nonepithelial expression of ERs in clinical (ERa) and research settings (ERb) over the years. In contrast to Knower et al. [5], we readily detect ERa in cultured breast fibroblasts from adjacent normal and cancer-associated breast tissue by qPCR. This is carried forward in observations by our group during the routine histopathological reporting of breast carcinoma, where the presence of ERa in stroma is seen in 60 % of all cases, although this can be very focal. Aware of reported issues with some ERa antibodies [6], we evaluated this further using three different antibodies directed against ERa, 1D5 (Dako), 6F11 (Novocastra) and SP1 (Ventana). Using all three antibodies four fifths of tumours expressed ERa in fibroblasts in concordant areas suggesting the signal is genuine (Fig. 1). The lack of ERa expression observed by Knower et al. [5] is thus surprising. We do agree that ERb gene expression is low, although it is usually detectable by qPCR. However, it is commonly found in breast fibroblasts by immunohistochemistry both total ERb [7] and specific isoforms (ERb1, -2 and -5) [8]. ERb is also subject to complex regulation [9], hence nonconcordance of gene and protein is anticipated. The nuclear receptors whose expression was modified in CAFs included RAR-related orphan receptor-a (ROR-a); thyroid hormone receptor-b (TR-b); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-c (PPAR-c) [5], which one may not immediately associate This letter to the editor refers to the article at 10.1007/s10549-0132716-6 and a rebuttal letter to this letter to the editor is available at 10. 1007/s10549-014-2837-6

Tumour suppressor gene function in carcinoma-associated fibroblasts: from tumour cells via EMT and back again?

Recent reports indicate that inactivation of the RB, TP53 or PTEN tumour suppressor genes is detected in tumour stroma of oropharyngeal, breast and other human cancers. Mouse models have validated the tumour-promoting effects of deleting Rb, Pten or p53 in fibroblasts that converts them from normal fibroblasts to carcinoma associated fibroblasts (CAFs). The tumour-promoting activity of CAFs in these contexts was associated with increased paracrine signaling to tumour cells through production of specific growth factors, chemokines and MMPs by CAFs. The conversion of NOFs into CAFs through acquisition of specific mutations, such as loss of tumour suppressors, or deregulated expression of microRNAs or key epigenetic events, can clearly occur independently of genetic and epigenetic changes in tumour cells but an alternative source of CAFs that is being reconsidered is that CAFs derive from the tumour cells by EMT. Recent mouse models employing lineage-tracing techniques have suggested that this can take place in vivo and the extent to which this is relevant more broadly is discussed.

Importance of stroma in breast cancer

Importance of stroma in breast cancer

English

English

Methods of Cell Propulsion through the Local Stroma in Breast Cancer.

In the normal breast, cellular structures change cyclically in response to ovarian hormones. Cell proliferation, apoptosis, invasion, and differentiation are integral processes that are precisely regulated. Normal epithelial cells depend on the formation of intercellular adhesion contacts to form a continuous sheet of stratifying cell layers that are attached to one and other horizontally and vertically. Cells migrate by extending membrane protrusions to explore the extracellular space locating their targets in a chemotactic manner. The formation of cell protrusions is driven by the assembly of actin filaments at the leading edge. Reorganisation is regulated by a highly integrated signalling cascade that transduces extracellular stimuli to the actin filaments. This signalling cascade is governed by GTPases which act as molecular switches leading to actin polymerisation and the formation of filopodia and lamellipodia. This process is linked to downstream molecules known collectively as WASP proteins, which, in the presence of cortactin, form a complex leading to nucleation and formation of branched filaments. In breast cancer, the cortactin is over expressed leading to increased cellular motility and invasiveness. This hugely complex and integrated signalling cascade transduces extracellular stimuli. There are multiple genes related to cell motility which are dysregulated in human breast cancers.

Markers of breast cancer stromal fibroblasts in the primary tumour site associated with lymph node metastasis: a systematic review including our case series

CAFs (cancer-associated fibroblasts), the most abundant cell type in breast cancer stroma, produce a plethora of chemokines, growth factors and ECM (extracellular matrix) proteins, that may contribute to dissemination and metastasis. Axillary nodes are the first metastatic site in breast cancer; however, to the present date, there is no consensus of which specific proteins, synthesized by CAFs, might be related with lymph node involvement. The purpose of this study was to perform a systematic review of CAF biomarkers associated with the presence of regional metastasis. PubMed was searched using the words: ‘breast cancer’ and ‘lymph node’ and fibroblast or stroma or microenvironment. After exclusions, eight studies evaluating biomarkers immunoexpression in CAFs and lymph node status were selected. Biomarkers evaluated in these studies may be divided in two groups, according to their ontology: extracellular matrix components [MMP13 (matrix metalloproteinase 13), TIMP2 (tissue inhibitor of metalloproteinases-2), THBS1 (thrombospondin 1), LGALS1 (lectin, galactoside-binding, soluble, 1)] and response to wounding [PDPN (podoplanin), PLAU (plasminogen activator, urokinase), PLAUR (plasminogen activator, urokinase receptor), CAV1 (caveolin 1), THBS1, LGALS1]. A positive expression of MMP13 and LGALS1 in CAFs was associated with enhanced OR (odds ratio) for regional metastasis. Contrariwise, CAV1 positive staining of fibroblasts was associated with decreased OR for nodal involvement. Expression of MMP13, PDPN and CAV1 was further tested in a new series of 65 samples of invasive ductal breast carcinomas by immunohistochemistry and no association between biomarkers expression in CAFs and nodal status was found. It was suggested that breast cancer subtypes may differentially affect CAFs behaviour. It would be interesting to evaluate the prognostic significance of these biomarkers in CAFs from different tumour types.

Fibroblast activation protein expression by stromal cells and tumor-associated macrophages in human breast cancer

Fibroblast activation protein (FAP) has long been known to be expressed in the stroma of breast cancer. However, very little is known if the magnitude of FAP expression within the stroma may have a prognostic value and reflect the heterogeneous biology of the tumor cell. An earlier study had suggested that stromal FAP expression in breast cancer was inversely proportional to prognosis. We, therefore, hypothesized that stromal FAP expression may correlate with clinicopathologic variables and may serve as an adjunct prognostic factor in breast cancer. We evaluated the expression of FAP in a panel of breast cancer tissues (n = 52) using a combination of immunostain analyses at the tissue and single-cell level using freshly frozen or freshly digested human breast tumor samples, respectively. Our results showed that FAP expression was abundantly expressed in the stroma across all breast cancer subtypes without significant correlation with clinicopathologic factors. We further identified a subset of FAP-positive (or FAP(+)) stromal cells that also expressed CD45, a pan-leukocyte marker. Using freshly dissociated human breast tumor specimens (n = 5), we demonstrated that some of these FAP(+)CD45(+) cells were CD11b(+)CD14(+)MHC-II(+), indicating that they were likely tumor-associated macrophages (TAMs). Although FAP(+)CD45(+) cells have been demonstrated in the mouse tumor stroma, our results demonstrating that human breast TAMs expressed FAP were novel and suggested that existing and future FAP-directed therapy may have dual-therapeutic benefits targeting both stromal mesenchymal cells and immune cells such as TAMs. More work is needed to explore the role of FAP as a potential targetable molecule in breast cancer treatment.