Project description:Tumor microenvironment coevolves with and simultaneously sustains cancer progression. Reactive fibroblasts found in prostate cancer (PCa), known as cancer associated fibroblasts (CAF), have been indeed shown to fuel tumor development and metastasis by mutually interacting with PCa cells. Little is known about the molecular mechanisms that lead to activation of CAFs from tissue-resident fibroblasts, circulating marrow-derived fibroblast progenitors or mesenchymal stem cells. Through integrated gene and microRNA expression profiling, here we showed that transcriptome of CAFs isolated from prostate tumors strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus confirming the capability of the cytokine to promote acquisition of an activated and cancer-promoting phenotype, and, for the first time, proving that IL6 is able per se to induce all the complex transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGFβ-related signatures, indicating that either signal, depending on the context, tumor stage and etiology, may concur to fibroblast activation. Our analyses also highlighted pathways relevant for induction of reactive stroma, including genes the role of which in fibroblast activation is still to be explored. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, in this study we provided insights on the molecular mechanisms driving fibroblast activation in prostate cancer, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. Tumor microenvironment coevolves with and simultaneously sustains cancer progression. Reactive fibroblasts found in prostate cancer (PCa), known as cancer associated fibroblasts (CAF), have been indeed shown to fuel tumor development and metastasis by mutually interacting with PCa cells. Little is known about the molecular mechanisms that lead to activation of CAFs from tissue-resident fibroblasts, circulating marrow-derived fibroblast progenitors or mesenchymal stem cells. Through integrated gene and microRNA expression profiling, here we showed that transcriptome of CAFs isolated from prostate tumors strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus confirming the capability of the cytokine to promote acquisition of an activated and cancer-promoting phenotype, and, for the first time, proving that IL6 is able per se to induce all the complex transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGFβ-related signatures, indicating that either signal, depending on the context, tumor stage and etiology, may concur to fibroblast activation. Our analyses also highlighted pathways relevant for induction of reactive stroma, including genes the role of which in fibroblast activation is still to be explored. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, in this study we provided insights on the molecular mechanisms driving fibroblast activation in prostate cancer, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. Tumor microenvironment coevolves with and simultaneously sustains cancer progression. Reactive fibroblasts found in prostate cancer (PCa), known as cancer associated fibroblasts (CAF), have been indeed shown to fuel tumor development and metastasis by mutually interacting with PCa cells. Little is known about the molecular mechanisms that lead to activation of CAFs from tissue-resident fibroblasts, circulating marrow-derived fibroblast progenitors or mesenchymal stem cells. Through integrated gene and microRNA expression profiling, here we showed that transcriptome of CAFs isolated from prostate tumors strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus confirming the capability of the cytokine to promote acquisition of an activated and cancer-promoting phenotype, and, for the first time, proving that IL6 is able per se to induce all the complex transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGFβ-related signatures, indicating that either signal, depending on the context, tumor stage and etiology, may concur to fibroblast activation. Our analyses also highlighted pathways relevant for induction of reactive stroma, including genes the role of which in fibroblast activation is still to be explored. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, in this study we provided insights on the molecular mechanisms driving fibroblast activation in prostate cancer, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. In the present study, patient derived-CAFs and HPFs activated in vitro with either TGFβ or IL6 were comparatively analyzed for gene and microRNA (miRNA) expression profiles, with the aim to define transcriptional pathways responsible for fibroblast activation and establish whether different subpopulations of CAFs may exist in PCa.

Project description:Tumor microenvironment coevolves with and simultaneously sustains cancer progression. Reactive fibroblasts found in prostate cancer (PCa), known as cancer associated fibroblasts (CAF), have been indeed shown to fuel tumor development and metastasis by mutually interacting with PCa cells. Little is known about the molecular mechanisms that lead to activation of CAFs from tissue-resident fibroblasts, circulating marrow-derived fibroblast progenitors or mesenchymal stem cells. Through integrated gene and microRNA expression profiling, here we showed that transcriptome of CAFs isolated from prostate tumors strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus confirming the capability of the cytokine to promote acquisition of an activated and cancer-promoting phenotype, and, for the first time, proving that IL6 is able per se to induce all the complex transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGF?-related signatures, indicating that either signal, depending on the context, tumor stage and etiology, may concur to fibroblast activation. Our analyses also highlighted pathways relevant for induction of reactive stroma, including genes the role of which in fibroblast activation is still to be explored. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, in this study we provided insights on the molecular mechanisms driving fibroblast activation in prostate cancer, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. Tumor microenvironment coevolves with and simultaneously sustains cancer progression. Reactive fibroblasts found in prostate cancer (PCa), known as cancer associated fibroblasts (CAF), have been indeed shown to fuel tumor development and metastasis by mutually interacting with PCa cells. Little is known about the molecular mechanisms that lead to activation of CAFs from tissue-resident fibroblasts, circulating marrow-derived fibroblast progenitors or mesenchymal stem cells. Through integrated gene and microRNA expression profiling, here we showed that transcriptome of CAFs isolated from prostate tumors strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus confirming the capability of the cytokine to promote acquisition of an activated and cancer-promoting phenotype, and, for the first time, proving that IL6 is able per se to induce all the complex transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGF?-related signatures, indicating that either signal, depending on the context, tumor stage and etiology, may concur to fibroblast activation. Our analyses also highlighted pathways relevant for induction of reactive stroma, including genes the role of which in fibroblast activation is still to be explored. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, in this study we provided insights on the molecular mechanisms driving fibroblast activation in prostate cancer, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. Tumor microenvironment coevolves with and simultaneously sustains cancer progression. Reactive fibroblasts found in prostate cancer (PCa), known as cancer associated fibroblasts (CAF), have been indeed shown to fuel tumor development and metastasis by mutually interacting with PCa cells. Little is known about the molecular mechanisms that lead to activation of CAFs from tissue-resident fibroblasts, circulating marrow-derived fibroblast progenitors or mesenchymal stem cells. Through integrated gene and microRNA expression profiling, here we showed that transcriptome of CAFs isolated from prostate tumors strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus confirming the capability of the cytokine to promote acquisition of an activated and cancer-promoting phenotype, and, for the first time, proving that IL6 is able per se to induce all the complex transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGF?-related signatures, indicating that either signal, depending on the context, tumor stage and etiology, may concur to fibroblast activation. Our analyses also highlighted pathways relevant for induction of reactive stroma, including genes the role of which in fibroblast activation is still to be explored. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression. Overall, in this study we provided insights on the molecular mechanisms driving fibroblast activation in prostate cancer, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment. In the present study, patient derived-CAFs and HPFs activated in vitro with either TGF? or IL6 were comparatively analyzed for gene and microRNA (miRNA) expression profiles, with the aim to define transcriptional pathways responsible for fibroblast activation and establish whether different subpopulations of CAFs may exist in PCa.

Project description:Prostate cancer (PCa) remains the malignancy with the highest morbidity in men (1). Although early-stage PCa usually causes no symptoms, cancer cells in prostate glands readily metastasize to bones, lymph nodes, lungs, and liver during PCa progression (2). Common strategies, including surgery, radiation therapy, and androgen-deprivation therapy, are promising treatments for early-stage PCa. However, advanced PCa still lacks effective therapeutic strategies (3-5). Thus, researchers are currently focused on inhibiting tumor progression to explore effective therapeutic strategies for PCa treatment (6, 7). Cancer-associated fibroblasts (CAFs), stromal cells derived from normal fibroblasts or epithelia, are one of the major cellular components in the tumor microenvironment (8, 9). Compared with normal fibroblasts, CAFs are activated with increased expression of key protein markers, such as α-SMA, FAP, vimentin and S100A4 protein (10, 11). In the tumor microenvironment, CAFs secrete various growth effectors or cytokines to the extracellular matrix and thereby promote tumor progression by directly enhancing cancer cell growth, angiogenic induction, the extracellular matrix modification, and tumor-promoting inflammatory mediation (11-14). Notably, CAFs could act as an immunosuppressive mediator for the formation of an immunosuppressive tumor environment by recruiting and activating multiple immune cells (15). As such, CAFs might be a potential target in anti-tumor immunotherapy (16, 17). Currently, increased research has focused on inhibiting the immunosuppressive function of prostate cancer-associated fibroblasts (prostate CAFs), which may suggest therapeutic strategies for PCa (18, 19). Polysaccharides from Lentinus edodes are known to exhibit potent anti-tumor and immunomodulatory functions. The anti-tumor mechanisms of L. edodes polysaccharides include regulating the innate immune system by mediating CAF function, preventing tumorigenesis, or directly killing tumor cells via cell cycle regulation or apoptotic induction (20-22). Our previous studies isolated a novel polysaccharide component (MPSSS) from L. edodes. We found the secretome of prostate CAFs treated with MPSSS could inhibit the proliferation of CD4+ and CD8+ T cells, which suggested that MPSSS could prevent the immunosuppressive function of prostate CAFs (22). However, although the secretome of MPSSS-treated prostate CAFs inhibit the proliferation of T cells, how the secretome of MPSSS-treated prostate CAFs affect PCa progression is still unclear. The secretome defines as all proteins secreted by the organism or living cells into the extracellular space, which consists of soluble proteins and extracellular vesicles (EVs) (23). The secretome of prostate CAFs pays vital roles in PCa tumor origination, progression (24, 25). Since EVs contains various molecules (proteins, RNA, DNA and lipid) (26), we speculate that soluble proteins and EVs of prostate CAFs might have the different influence on PCa cells. In this study, the secretome of prostate CAFs untreated/treated with MPSSS were separated into the high molecular weight secretome (>100 kD) and the low molecular weight secretome (3-100 kD) using 100 kD and 3kD MWCO memberane filtration to narrow down the range of active components. The high molecular weight secretome contained extracellular vesicles (EVs) and large soluble proteins, while the low molecular weight secretome contained the small soluble proteins. The secretome of prostate CAFs untreated/treated with MPSSS were used to explore the underlying function and molecular mechanism using quantitative proteomics and multiple biochemical approaches.

Project description:Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging, hence sampling circulating tumor cells (CTCs) may reveal drug resistance mechanisms. We established single cell RNA-sequencing profiles of 77 intact CTCs isolated from 13 patients (mean 6 CTCs/patient) using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing on AR inhibitor, compared with untreated cases indicates activation of noncanonical Wnt signaling (P=0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, while its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure. A total of 221 single candidate prostate CTCs were isolated from 18 patients with metastatic prostate cancer and 4 patients with localized prostate cancer. Of these, 133 cells (60%) had RNA of sufficient quality for amplification and next generation RNA sequencing, and 122 (55%) had >100,000 uniquely aligned sequencing reads. In addition to candidate CTCs, we also obtained comprehensive transcriptomes for 12 bulk primary prostate cancers (macrodissected for >70% tumor content), 30 single cells derived from four different prostate cancer cell lines, and 5 patient-derived leukocyte controls. The leukocytes were readily distinguished by their expression of hematopoietic lineage markers and served to exclude any CTCs with potentially contaminating signals. Strict expression thresholds were used to define lineage-confirmed CTCs, scored by prostate lineage-specific genes (PSA, PSMA, AMACR, AR) and standard epithelial markers (KRT7, KRT8, KRT18, KRT19, EpCAM). 28 cells were excluded given the presence of leukocyte transcripts suggestive of cellular contamination or misidentification during selection, and 17 cells were excluded given low expression of both prostate lineage-specific genes and 5 standard epithelial markers. The remaining 77 cells, defined as lineage-confirmed CTCs, displayed expression of either prostate lineage-specific or epithelial genes, and low expression of leukocyte-specific genes, consistent with their tumor of origin.

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Integrated miRNA and gene expression analysis of prostate cancer associated fibroblasts supports a prominent role for interleukin-6 in fibroblast activation

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