Arousal of cancer-associated stroma: overexpression of palladin activates fibroblasts to promote tumor invasion

Background

Cancer-associated fibroblasts, comprised of activated fibroblasts or myofibroblasts, are found in the stroma surrounding solid tumors. These myofibroblasts promote invasion and metastasis of cancer cells. Mechanisms regulating the activation of the fibroblasts and the initiation of invasive tumorigenesis are of great interest. Upregulation of the cytoskeletal protein, palladin, has been detected in the stromal myofibroblasts surrounding many solid cancers and in expression screens for genes involved in invasion. Using a pancreatic cancer model, we investigated the functional consequence of overexpression of exogenous palladin in normal fibroblasts in vitro and its effect on the early stages of tumor invasion.

Principal Findings

Palladin expression in stromal fibroblasts occurs very early in tumorigenesis. In vivo, concordant expression of palladin and the myofibroblast marker, alpha smooth muscle actin (α-SMA), occurs early at the dysplastic stages in peri-tumoral stroma and progressively increases in pancreatic tumorigenesis. In vitro introduction of exogenous 90 kD palladin into normal human dermal fibroblasts (HDFs) induces activation of stromal fibroblasts into myofibroblasts as marked by induction of α-SMA and vimentin, and through the physical change of cell morphology. Moreover, palladin expression in the fibroblasts enhances cellular migration, invasion through the extracellular matrix, and creation of tunnels through which cancer cells can follow. The fibroblast invasion and creation of tunnels results from the development of invadopodia-like cellular protrusions which express invadopodia proteins and proteolytic enzymes. Palladin expression in fibroblasts is triggered by the co-culture of normal fibroblasts with k-ras-expressing epithelial cells.

Conclusions

Overall, palladin expression can impart myofibroblast properties, in turn promoting the invasive potential of these peri-tumoral cells with invadopodia-driven degradation of extracellular matrix. Palladin expression in fibroblasts can be triggered by k-ras expression in adjacent epithelial cells. This data supports a model whereby palladin-activated fibroblasts facilitate stromal-dependent metastasis and outgrowth of tumorigenic epithelium.     

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle -actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

Ets2 in Tumor Fibroblasts Promotes Angiogenesis in Breast Cancer

Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.     

Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts in coculture

Summary In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.     

Catalytically inactive human cathepsin D triggers fibroblast invasive growth

The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial-fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D-deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras-MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.     

Direct contacts with colon cancer cells regulate the differentiation of bone marrow mesenchymal stem cells into tumor associated fibroblasts

Tumor–stroma interactions are referred to as essential events in tumor progression. There has been growing attention that bone marrow-derived mesenchymal stem cells (BMSCs) can travel to tumor stroma, where they differentiate into tumor-associated fibroblast (TAF)-like cells, a predominant tumor-promoting stromal cell. However, little is definitively known about the contributors for this transition. Here, using an in vitro direct co-culture model of colon cancer cells and BMSCs, we identify that colon cancer cells can induce adjoining BMSCs to exhibit the typical characteristic of TAFs, with increased expression of α-smooth muscle actin (α-SMA). Importantly, the present data also reveals that activated Notch signaling mediates transformation of BMSCs to TAFs through the downstream TGF-β/Smad signaling pathway.     

Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting

Background

Important support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.

Methods and Findings

We used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.

Conclusions

This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.     

A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation

The objective of our study was to establish spheroid cocultures as a valid 3-D in vitro model mimicking tumor-fibroblast interactions in scirrhous breast tumors. The experimental setup was designed to verify if in cocultures (a) adherence and migration reflect the invasive potential of breast tumor cells, (b) breast tumor cells induce tumor-associated fibroblast differentiation, and (c) tumor-derived fibroblasts better reflect the in vivo situation than normal skin fibroblasts. Only one (SK-BR-3) out of five tumor cell types showed extensive fibroblast infiltration, MCF-7 cells frequently invaded fibroblast spheroids; BT474, T47D, and ZR-75-1 were noninvasive. While tumor cell invasion was independent of fibroblast origin, tumor-associated myofibroblast differentiation defined by alpha-SMA expression was demonstrated for tumor-derived but not normal skin fibroblasts in coculture indicating that (a) tumor cell invasion and myofibroblast differentiation are autonomous processes and (b) cocultures with tumor-derived fibroblasts resemble advanced stages of desmoplastic carcinomas while cocultures with normal skin fibroblasts rather reflect the early tumor development. The latter is also implied by fibroblast-associated alterations in tumor cell morphology and ECM distribution in the system. By using RNA arbitrarily primed PCR and cells isolated from cocultures by fluorescence-activated and magnetic cell separation, peripheral myelin protein PMP22/SR13 has been identified as a novel candidate with potential relevance in the interaction between tumor cell and normal fibroblast since PMP22 mRNA was significantly reduced in normal skin fibroblasts in coculture with BT474 cells.     

CD105 Expression on CD34-Negative Spindle-Shaped Stromal Cells of Primary Tumor Is an Unfavorable Prognostic Marker in Early Breast Cancer Patients

Several studies have confirmed that the breast tumor microenvironment drives cancer progression and metastatic development. The aim of our research was to investigate the prognostic significance of the breast tumor microenvironment in untreated early breast cancer patients. Therefore, we analyzed the association of the expression of α-SMA, FSP, CD105 and CD146 in CD34-negative spindle-shaped stromal cells, not associated with the vasculature, in primary breast tumors with classical prognostic marker levels, metastatic recurrence, local relapse, disease-free survival, metastasis-free survival and the overall survival of patients. In the same way, we evaluated the association of the amount of intra-tumor stroma, fibroblasts, collagen deposition, lymphocytic infiltration and myxoid changes in these samples with the clinical-pathological data previously described. This study is the first to demonstrate the high CD105 expression in this stromal cell type as a possible independent marker of unfavorable prognosis in early breast cancer patients. Our study suggests that this new finding can be useful prognostic marker in the clinical-pathological routine.     

Experimental generation of carcinoma-associated fibroblasts (CAFs) from human mammary fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes (1-3). The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression(4,5). These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues (6). The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis (7-10). CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance (11). We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.     

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin^cpdm), and mice with a stromal (S100a4‐Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin^cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin^cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin^cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.     

Imatinib mesylate inhibits proliferation and exerts an antifibrotic effect in human breast stroma fibroblasts

Tumor stroma plays an important role in cancer development. In a variety of tumors, such as breast carcinomas, a desmoplastic response, characterized by stromal fibroblast and collagen accumulation, is observed having synergistic effects on tumor progression. However, the effect of known anticancer drugs on stromal cells has not been thoroughly investigated. Imatinib mesylate is a selective inhibitor of several protein tyrosine kinases, including the receptor of platelet-derived growth factor, an important mediator of desmoplasia. Recently, we have shown that imatinib inhibits the growth and invasiveness of human epithelial breast cancer cells. Here, we studied the effect of imatinib on the proliferation and collagen accumulation in breast stromal fibroblasts. We have shown that it blocks the activation of the extracellular signal-regulated kinase and Akt signaling pathways and up-regulates cyclin-dependent kinase inhibitor p21(WAF1), leading to the inhibition of fibroblast proliferation, by arresting them at the G(0)/G(1) phase of the cell cycle. Imatinib inhibits more potently the platelet-derived growth factor-mediated stimulation of breast fibroblast proliferation. By using specific inhibitors, we have found that this is due to the inhibition of the Akt pathway. In addition, imatinib inhibits fibroblast-mediated collagen accumulation. Conventional and quantitative PCR analysis, as well as gelatin zymography, indicates that this is due to the down-regulation of mRNA synthesis of collagen I and collagen III-the main collagen types in breast stroma-and not to the up-regulation or activation of collagenases matrix metalloproteinase 2 and matrix metalloproteinase 9. These data indicate that imatinib has an antifibrotic effect on human breast stromal fibroblasts that may inhibit desmoplastic reaction and thus tumor progression.     

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Background information. Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer‐associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT‐PCR (quantitative real‐time PCR) and immunohistochemistry. Results. We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin‐like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. Conclusions. Our observations indicate the pro‐stem cell activity of cancer‐associated fibroblasts and underline the role of epithelial—mesenchymal interaction in tumour biology.     

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: Implications for breast cancer and DCIS therapy with autophagy inhibitors

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating “synthetic” cancer associated fibroblasts (CAFs). More specifically, these “synthetic” CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].