Human Cancer-associated Fibroblasts Research Articles

Cathepsin D acts as an essential mediator to promote malignancy of benign prostatic epithelium.

Stromal-epithelial interactions are important in both development and prostate cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. We investigated whether stromal cathepsin D (CathD) overexpression affects prostate tumorigenesis through a paracrine mechanism. Normal prostate fibroblasts (NPF) were retrovirally transduced to overexpress cyclin D1 (CD1) and were designated NPF(CD1) . Cathepsin D expression was knocked down using shRNA in cancer associated fibroblasts (CAF) and NPF(CD1) . We analyzed these stromal cell lines using immunohistochemistry, Western blot, and tissue recombination. An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue. Overexpression of CD1 in normal prostate fibroblasts (NPF(CD1) ) produced a phenotype similar to, but more moderate than, CAF in a tissue recombination model. Knockdown studies revealed that CathD is required for NPF(CD1) motility and invasive growth in vitro. BPH-1 cell proliferation was found to be induced when cultured with NPF(CD1) conditioned medium, this effect was inhibited when CathD was knocked down in NPF(CD1) cells. Overexpression of CathD in prostate stromal cells induced malignancy in adjacent epithelium, and this transformation was inhibited when stromal CathD expression was knocked down in CAF. The study presented here demonstrates increased CathD expression is seen in human CAF. The upregulation of CD1 results in concomitant increases in CathD expression. Elevated CathD expression in the stroma contributes to tumor promotion.

Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

Human Epithelial Basal Cells Are Cells of Origin of Prostate Cancer, Independent of CD133 Status

Normal prostatic epithelium is composed of basal and luminal cells. Prostate cancer can be initiated in both benign basal and luminal stem cells, but because basal cell markers are not expressed in patient tumors, the former result was unexpected. Since the cells of origin of prostate cancer are important therapeutic targets, we sought to provide further proof that basal stem cells have tumorigenic potential. Prostatic basal cells were enriched based on α2β1integrin(hi) expression and further enriched for stem cells using CD133 in nontumorigenic BPH-1 cells. Human embryonic stem cells (hESCs) were also used as a source of normal stem cells. To test their tumorigenicity, we used two alternate stromal-based approaches; (a) recombination with human cancer-associated fibroblasts (CAFs) or (b) recombination with embryonic stroma (urogenital mesenchyme) and treated host mice with testosterone and 17β-estradiol. Enriched α2β1integrin(hi) basal cells from BPH-1 cells resulted in malignant tumor formation using both assays of tumorigenicity. Surprisingly, the tumorigenic potential did not reside in the CD133(+) stem cells but was consistently observed in the CD133(-) population. CAFs also failed to induce prostatic tumors from hESCs. These data confirmed that benign human basal cells include cells of origin of prostate cancer and reinforced their importance as therapeutic targets. In addition, our data suggested that the more proliferative CD133(-) basal cells are more susceptible to tumorigenesis compared to the CD133(+)-enriched stem cells. These findings challenge the current dogma that normal stem cells and cells of origin of cancer are the same cell type(s).

Abstract 54: Cancer-associated fibroblasts induce epithelial-mesenchymal-transition and stemness in non-small-cell lung cancer

Abstract The tumor microenvironment is composed of different types of stromal cells and a key component of tumor progression. It has been hypothesized that these stromal components are functionally organized to promote the survival of cancer cells. A specific subset of stromal cells, termed cancer-associated fibroblasts (CAFs), secretes factors that promote tumorigensis. To investigate whether CAFs constitute a specific microenvironment conductive for lung cancer progression, we performed in vitro and in vivo experiments using CAFs derived from primary lung cancer in conjunction with the human lung adenocaricinoma cell lines. We first showed that conditioned medium (CM) from human CAFs was biologically active. When human lung adenocarcinoma cell lines (A549 and NCI-H358) were cultured with CM from CAFs, epithelial cells became spindle and scattered as well as decreased cell surface E-cadherin. Further CM increased N-cadherin and vimentin expression, which is a hallmark of an epithelial-mesenchymal-transition (EMT). Coculture experiments using traswell plates with inserts of pore size 3 μm also showed EMT change in cancer cells. CM led cancer cells to enhance expression of stem cell markers such as CD44 and CD133 mRNA levels by ten times. It also triggered the acquisitions of cancer stemness such the ability of resistance to cisplatin or gefitinib as well as sphere formation. These phenotype changes associated with EMT and stemness were inhibited by the addition of TGF-β inhibitor, SB431542 (10 μM). The s.c. coinjection of human lung adenocarcinoma cell lines and human CAFs to the mice result in a high rate of tumor formation compared with the injection of these cell lines without CAFs. A decreased expression of epithelial markers and upregulation of angiogenesis and proliferative index were detected in specimens from mice with coinjection of these cells. TGF β inhibitor attenuated the tumor formation enhanced by coinjection with CAFs. The compound was injected i.p. at 0.2 mg/kg (1 per day, 5 per week for 2 weeks) after coinjection. These results indicate that the paracrine interplay between CAFs and cancer cells mediated by TGF-β signaling leads to an EMT driven gain of cancer stem cell properties and suggest that the CAFs contribute a specific niche for tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 54. doi:1538-7445.AM2012-54

Leptin Mediates Tumor–Stromal Interactions That Promote the Invasive Growth of Breast Cancer Cells

Obesity confers risks to cancer development and progression but the mechanisms underlying these risks remain unclear. In this study, we identify a role for the obesity cytokine leptin, which has been implicated previously in breast cancer development, as a determinant for the tumor-promoting activity of cancer-associated fibroblasts (CAF) in both wild-type (WT) and K303R mutant estrogen receptor-α (ERα)-expressing breast cancer cells. Human CAFs stimulated a greater increase in the proliferation and migration of breast cancer cells expressing the K303R-ERα hyperactive receptor than WT-ERα-expressing cells. A concomitant increase was seen in leptin receptor isoform expression and activation of the leptin signaling pathway in cells expressing K303R-ERα compared with WT-ERα, correlating with leptin effects on cell growth, motility, and invasiveness in mutant cells. Epidermal growth factor and other factors secreted by K303R-ERα cells stimulated CAF proliferation, migration, and subsequent leptin secretion. Moreover, K303R-ERα expression generated a leptin hypersensitive phenotype in vivo. Together, our results reveal a bidirectional cross-talk between breast cancer cells and "educated" CAFs that drives tumor progression via leptin signaling. In elucidating a mechanism that connects obesity and cancer, these findings reinforce the concept that blocking cancer-stromal cell communication may represent an effective strategy for targeted therapy of breast cancer.

Adenovirus i-Leader Truncation Bioselected Against Cancer-associated Fibroblasts to Overcome Tumor Stromal Barriers

Tumor-associated stromal cells constitute a major hurdle in the antitumor efficacy with oncolytic adenoviruses. To overcome this biological barrier, an in vitro bioselection of a mutagenized AdwtRGD stock in human cancer-associated fibroblasts (CAFs) was performed. Several rounds of harvest at early cytopathic effect (CPE) followed by plaque isolation led us to identify one mutant with large plaque phenotype, enhanced release in CAFs and enhanced cytotoxicity in CAF and several tumor cell lines. Whole genome sequencing and functional mapping identified the truncation of the last 17 amino acids in C-terminal end of the i-leader protein as the mutation responsible for this phenotype. Similar mutations have been previously isolated in two independent bioselection processes in tumor cell lines. Importantly, our results establish the enhanced antitumor activity in vivo of the i-leader C-terminal truncated mutants, especially in a desmotic fibroblast-embedded lung carcinoma model in mice. These results indicate that the i-leader truncation represents a promising trait to improve virotherapy with oncolytic adenoviruses.

Breast Cancer Cells Induce Cancer-Associated Fibroblasts to Secrete Hepatocyte Growth Factor to Enhance Breast Tumorigenesis

It has been well documented that microenvironment consisting of stroma affects breast cancer progression. However, the mechanisms by which cancer cells and fibroblasts, the major cell type in stroma, interact with each other during tumor development remains to be elucidated. Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients. The expression level of hepatocyte growth factor (HGF) in these fibroblasts was positively correlated with their ability to enhance breast tumorigenesis in mice. Deprivation of HGF using a neutralizing antibody reduced CAF-mediated colony formation of human breast cancer cells, indicating that CAFs enhanced cancer cell colony formation mainly through HGF secretion. Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity. The newly gained ability of these “educated” NAFs became irreversible after continuing this process till fourth passage. These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling.

The role of miR-31 and its target gene SATB2 in cancer-associated fibroblasts

It is well established that there is a dynamic relationship between the expanding tumor and the host surrounding tissue. Cancer-associated fibroblasts (CAFs), the most common cellular population found in the tumor microenvironment, supporting tumor growth and dissemination. Here, we set out to determine the factors that may be involved in dramatic alteration of gene expression pattern in CAFs, focusing on microRNA and transcriptional regulators. We established matched pairs of human CAFs isolated from endometrial cancer and normal endometrial fibroblasts. MicroRNA and mRNA analyses identified differential expression of 11 microRNAs, with miR-31 being the most downregulated microRNA in CAFs (p=0.007). We examined several putative miR-31 target genes identified by microarray analysis and demonstrated that miR-31 directly targets the homeobox gene SATB2, which is responsible for chromatin remodeling and regulation of gene expression, and was significantly elevated in CAFs. The functional relevance of miR-31 and SATB2 were tested in in vitro models of endometrial cancer. Overexpression of miR-31 significantly impaired the ability of CAFs to stimulate tumor cell migration and invasion, without affecting tumor cell proliferation. Genetic manipulation of SATB2 levels in normal fibroblasts or CAFs showed that, reciprocally to miR-31, SATB2 increased tumor cell migration and invasion, while knock-down of endogenous SATB2 in CAFs reversed this phenotype. Introduction of SATB2 into normal fibroblasts stimulated expression of a number of genes involved in cell invasion, migration and scattering. These findings provide new insights into tumor-stroma interaction and document that miR

Caveolin-1 −/− Null Mammary Stromal Fibroblasts Share Characteristics with Human Breast Cancer-Associated Fibroblasts

Recently, we reported that human breast cancer-associated fibroblasts show functional inactivation of the retinoblastoma (RB) tumor suppressor and down-regulation of caveolin-1 (Cav-1) protein expression. However, it remains unknown whether loss of Cav-1 is sufficient to confer functional RB inactivation in mammary fibroblasts. To establish a direct cause-and-effect relationship, mammary stromal fibroblasts (MSFs) were prepared from Cav-1(-/-) null mice and subjected to phenotypic analysis. Here, we provide evidence that Cav-1(-/-) MSFs share many characteristics with human cancer-associated fibroblasts. The Cav-1(-/-) MSF transcriptome significantly overlaps with human cancer-associated fibroblasts; both show a nearly identical profile of RB/E2F-regulated genes that are up-regulated, which is consistent with RB inactivation. This Cav-1(-/-) MSF gene signature is predictive of poor clinical outcome in breast cancer patients treated with tamoxifen. Consistent with these findings, Cav-1(-/-) MSFs show RB hyperphosphorylation and the up-regulation of estrogen receptor co-activator genes. We also evaluated the paracrine effects of "conditioned media" prepared from Cav-1(-/-) MSFs on wild-type mammary epithelia. Our results indicate that Cav-1(-/-) MSF "conditioned media" is sufficient to induce an epithelial-mesenchymal transition, indicative of an invasive phenotype. Proteomic analysis of this "conditioned media" reveals increased levels of proliferative/angiogenic growth factors. Consistent with these findings, Cav-1(-/-) MSFs are able to undergo endothelial-like transdifferentiation. Thus, these results have important implications for understanding the role of cancer-associated fibroblasts and RB inactivation in promoting tumor angiogenesis.

Cancer cells suppress p53 in adjacent fibroblasts

The p53 tumor suppressor serves as a crucial barrier against cancer development. In tumor cells and their progenitors, p53 suppresses cancer in a cell-autonomous manner. However, p53 also possesses non-cell-autonomous activities. For example, p53 of stromal fibroblasts can modulate the spectrum of proteins secreted by these cells, rendering their microenvironment less supportive of the survival and spread of adjacent tumor cells. We now report that epithelial tumor cells can suppress p53 induction in neighboring fibroblasts, an effect reproducible by tumor cell-conditioned medium. The ability to suppress fibroblast p53 activation is acquired by epithelial cells in the course of neoplastic transformation. Specifically, stable transduction of immortalized epithelial cells by mutant H-Ras and p53-specific short inhibitory RNA endows them with the ability to quench fibroblast p53 induction. Importantly, human cancer-associated fibroblasts are more susceptible to this suppression than normal fibroblasts. These findings underscore a mechanism whereby epithelial cancer cells may overcome the non-cell-autonomous tumor suppressor function of p53 in stromal fibroblasts.