Aldehyde dehydrogenase 3B2 promotes the proliferation and invasion of cholangiocarcinoma by increasing Integrin Beta 1 expression

Aldehyde dehydrogenases (ALDHs) play an essential role in regulating malignant tumor progression; however, their role in cholangiocarcinoma (CCA) has not been elucidated. We analyzed the expression of ALDHs in 8 paired tumor and peritumor perihilar cholangiocarcinoma (pCCA) tissues and found that ALDH3B1 and ALDH3B2 were upregulated in tumor tissues. Further survival analysis in intrahepatic cholangiocarcinoma (iCCA, n = 27), pCCA (n = 87) and distal cholangiocarcinoma (dCCA, n = 80) cohorts have revealed that ALDH3B2 was a prognostic factor of CCA and was an independent prognostic factor of iCCA and pCCA. ALDH3B2 expression was associated with serum CEA in iCCA and dCCA, associated with tumor T stage, M stage, neural invasion and serum CA19-9 in pCCA. In two cholangiocarcinoma cell lines, overexpression of ALDH3B2 promoted cell proliferation and clone formation by promoting the G1/S phase transition. Knockdown of ALDH3B2 inhibited cell migration, invasion, and EMT in vitro, and restrained tumor metastasis in vivo. Patients with high expression of ALDH3B2 also have high expression of ITGB1 in iCCA, pCCA, and dCCA at both mRNA and protein levels. Knockdown of ALDH3B2 downregulated the expression of ITGB1 and inhibited the phosphorylation level of c-Jun, p38, and ERK. Meanwhile, knockdown of ITGB1 inhibited the promoting effect of ALDH3B2 overexpression on cell proliferation, migration, and invasion. ITGB1 is also a prognostic factor of iCCA, pCCA, and dCCA and double-positive expression of ITGB1 and ALDH3B2 exhibits better performance in predicting patient prognosis. In conclusion, ALDH3B2 promotes tumor proliferation and metastasis in CCA by regulating the expression of ITGB1 and upregulating its downstream signaling pathway. The double-positive expression of ITGB1 and ALDH3B2 serves as a better prognostic biomarker of CCA.Subject terms: Prognostic markers, Bile duct cancer     

MiR-210 in exosomes derived from CAFs promotes non-small cell lung cancer migration and invasion through PTEN/PI3K/AKT pathway

ObjectiveCancer-associated fibroblasts (CAFs) function as a crucial factor in tumor progression by carrying exosomes to neighboring cells. This study was assigned to expound the underlying mechanism of CAFs-derived exosomal miR-210 in non-small cell lung cancer (NSCLC) progression.MethodCAFs and normal fibroblasts (NFs) were isolated and identified. Exosomes secreted from CAFs and NFs were isolated to analyze their effects on tumor volume and epithelial-mesenchymal transition (EMT). Exosomal miR-210 expression level was measured. The effects of exosomal miR-210 and UPF1 on cell viability, EMT, PTEN/PI3K/AKT signal pathway were determined. Dual-luciferase reporter gene assay was utilized to validate the binding of UPF1 to miR-210.ResultsCAFs-derived exosomes (CAFs-exo) were successfully extracted and proven to be uptake by lung cancer cells. Up-regulated expression level of miR-210 was found in CAFs-exo, which was then proved to enhance cell migration, proliferation, invasion abilities and EMT in NSCLC cells. Overexpression of miR-210 can also inhibit UPF1 and PTEN, but activate the PTEN/PI3K/AKT pathway. UPF1 was a target gene of miR-210. MiR-210 can up-regulate UPF1 expression level to activate PTEN/PI3K/AKT pathway.ConclusionMiR-210 secreted by CAFs-exo could promote EMT by targeting UPF1 and activating PTEN/PI3K/AKT pathway, thereby promoting NSCLC migration and invasion.     

Loss of exosomal miR-148b from cancer-associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis

Cancer-associated fibroblasts (CAFs) play crucial roles in tumor progression, given the dependence of cancer cells on stromal support. Therefore, understanding how CAFs communicate with endometrial cancer cell in tumor environment is important for endometrial cancer therapy. Exosomes, which contain proteins and noncoding RNA, are identified as an important mediator of cell–cell communication. However, the function of exosomes in endometrial cancer metastasis remains poorly understood. In the current study we found that CAF-derived exosomes significantly promoted endometrial cancer cell invasion comparing to those from normal fibroblasts (NFs). We identified a significant decrease of miR-148b in CAFs and CAFs-derived exosomes. By exogenously transfect microRNAs, we demonstrated that miR-148b could be transferred from CAFs to endometrial cancer cell through exosomes. In vitro and in vivo studies further revealed that miR-148b functioned as a tumor suppressor by directly binding to its downstream target gene, DNMT1 to suppress endometrial cancer metastasis. In endometrial cancer DNMT1 presented a potential role in enhancing cancer cell metastasis by inducing epithelial–mesenchymal transition (EMT). Therefore, downregulated miR-148b induced EMT of endometrial cancer cell as a result of relieving the suppression of DNMT1. Taken together, these results suggest that CAFs-mediated endometrial cancer progression is partially related to the loss of miR-148b in the exosomes of CAFs and promoting the transfer of stromal cell-derived miR-148b might be a potential treatment to prevent endometrial cancer progression.     

MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer

Cancer-associated fibroblasts (CAFs) promote tumorigenesis, growth, invasion and metastasis of cancer, whereas normal fibroblasts (NFs) are thought to suppress tumor progression. Little is known about miRNAs expression differences between CAFs and NFs or the patient-to-patient variability in miRNAs expression in breast cancer. We established primary cultures of CAFs and paired NFs from six resected breast tumor tissues that had not previously received radiotherapy or chemotherapy treatment and analyzed with miRNAs microarrays. The array data were analyzed using paired SAM t-test and filtered according to α and q values. Pathway analysis was conducted using DAVID v6.7. We identified 11 dysregulated miRNAs in CAFs: three were up-regulated (miR-221-5p, miR-31-3p, miR-221-3p), while eight were down-regulated (miR-205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p, let-7g, miR-26b). Their target genes are known to affect cell differentiation, adhesion, migration, proliferation, secretion and cell-cell interaction. By our knowledge it is firstly identify the expression profiles of miRNAs between CAFs and NFs and revealed their regulation on the associated signaling pathways.     

Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.Subject terms: Tumour biomarkers, Cancer     

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.     

Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts

It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma.Tumorigenesis is not considered anymore a tumor cell-autonomous mechanism triggered by accumulation of somatic aberrations, but fostered by a two-way interaction between cancer cells and the surrounding microenvironment.Cancer cells are indeed integrated in a biologically complex stroma, composed of different cell types (such as immune system components, endothelial cells, fibroblasts and adipocytes) as well as extracellular matrix (ECM), which originates the heterogeneity of the tumor microenvironment (TME).¹ It is known that a permissive TME has a key role in tumorigenesis.Fibroblasts, which represent the majority of the stromal cells, are very active in the ECM synthesis, regulation of inflammation and wound healing.² Even though the communication between cancer cells and fibroblasts has been extensively described,³ it is still currently unclear how this interaction promotes the activation of quiescent fibroblasts in cancer-associated fibroblasts (CAFs). It has been reported that breast carcinoma-associated stroma differs from its paired normal in deregulated expression of cytokines, ECM molecules and metalloproteinases.^{4, 5}Breast cancer is the leading cause of cancer-related deaths in women.⁶ Clinically, this heterogeneous disease is categorized into four major molecular subtypes: luminal-A, luminal-B, human epidermal growth factor receptor 2 (HER2) overexpressing and triple-negative/basal-like. Triple-negative breast cancer (TNBC) constitutes approximately 15–20% of all breast cancer cases, with the worst outcome of all subtypes.⁷In breast cancer, the biological characteristics and genetic heterogeneity between CAFs and their paired normal fibroblasts (NFs) have been described.^{8, 9} Breast CAFs are characterized by stronger ability in proliferation and cell motility in comparison with NFs and, consistently with this biological behavior, gene expression profiling showed the abnormal regulation of key signaling pathways as cell adhesion and secreting factors in CAFs.¹⁰MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that play an important role in various biological processes.¹¹ Their extracellular presence as the major RNA component of exosomes suggests an internalization process by TME cells, thus mediating the cancer–host communication and participating in cancer metastasis by adapting the cell niches.¹² To date, little is known about miRNA expression differences between CAFs and NFs. Array data of primary cultures of CAFs versus their paired NFs from resected breast tumor tissues identified 11 dysregulated miRNAs, and their predicted target genes resulted mainly related to adhesion, migration, secretion and cell–cell interaction.¹³ A set of three miRNAs has been described to be involved in reprogramming NFs to CAFs in ovarian cancer¹⁴ and, very recently, miR-200s were found to contribute to breast cancer cell invasion through CAF activation and ECM remodeling.¹⁵In the present work, our attention focused on miR-9 as a possible player in the cross-talk between breast cancer cells and stroma. Numerous evidence supports this hypothesis: miR-9 has been described as metastamiR in breast cancer and it resulted markedly upregulated in breast cancer cells compared with normal mammary tissues.¹⁶ MiR-9 directly targets E-cadherin (CDH1) leading to increase cancer cell motility and invasiveness.¹⁷ Even more interestingly, miR-9 was found to be secreted by different human tumor cell lines, packaged into microvesicles and directly delivered to endothelial cells where it is able to promote migration and neovascularization activating JACK–STAT pathway. These observations suggest that tumor-secreted miRNAs can be involved in intercellular communication.¹⁸ Moreover, recent data showed that miR-9 overexpression is associated with epithelial–mesenchymal transition and poor prognosis in breast cancer, leading to its possible use as a biomarker for cancer progression and a target for treatment.¹⁹Our data revealed a higher expression of miR-9 in primary triple-negative breast CAFs versus NFs isolated from patients. Cell motility assays of immortalized NFs overexpressing miR-9 and CAFs where the miRNA was inhibited showed miR-9''s ability to affect the fibroblast behavior. Furthermore, tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake resulted in enhanced cell motility. Gene expression profiles allowed us to identify a subgroup of molecules differentially expressed in NFs overexpressing miR-9 (NFs/miR-9) mainly involved in cell motility pathways and ECM remodeling. Moreover, miR-9-mediated downmodulation of its known target CDH1 in breast cancer cells cultured in conditioned medium from NFs/miR-9 indicated that miR-9 is also released by fibroblasts and transferred to tumor cells, and provided details regarding the biological mechanisms that could explain both the stronger motility and invasiveness of breast cancer cells observed in vitro, and the improved in vivo growth following co-injection with NFs/miR-9.     

Loss of Numb promotes hepatic progenitor expansion and intrahepatic cholangiocarcinoma by enhancing Notch signaling

Numb, a stem cell fate determinant, acts as a tumor suppressor and is closely related to a wide variety of malignancies. Intrahepatic cholangiocarcinoma (iCCA) originates from hepatic progenitors (HPCs); however, the role of Numb in HPC malignant transformation and iCCA development is still unclear. A retrospective cohort study indicated that Numb was frequently decreased in tumor tissues and suggests poor prognosis in iCCA patients. Consistently, in a chemically induced iCCA mouse model, Numb was downregulated in tumor cells compared to normal cholangiocytes. In diet-induced chronic liver injury mouse models, Numb ablation significantly promoted histological impairment, HPC expansion, and tumorigenesis. Similarly, Numb silencing in cultured iCCA cells enhanced cell spheroid growth, invasion, metastasis, and the expression of stem cell markers. Mechanistically, Numb was found to bind to the Notch intracellular domain (NICD), and Numb ablation promoted Notch signaling; this effect was reversed when Notch signaling was blocked by γ-secretase inhibitor treatment. Our results suggested that loss of Numb plays an important role in promoting HPC expansion, HPC malignant transformation, and, ultimately, iCCA development in chronically injured livers. Therapies targeting suppressed Numb are promising for the treatment of iCCA.Subject terms: Cancer, Liver cancer     

Cancer-Associated Fibroblasts in a Human HEp-2 Established Laryngeal Xenografted Tumor Are Not Derived from Cancer Cells through Epithelial-Mesenchymal Transition,Phenotypically Activated but Karyotypically Normal

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model.     

Stromal miR-200s contribute to breast cancer cell invasion through CAF activation and ECM remodeling

The activation of cancer-associated fibroblasts (CAFs) is a key event in tumor progression, and alternative extracellular matrix (ECM) proteins derived from CAFs induce ECM remodeling and cancer cell invasion. Here we found that miR-200 s, which are generally downregulated in activated CAFs in breast cancer tissues and in normal fibroblasts (NFs) activated by breast cancer cells, are direct mediators of NF reprogramming into CAFs and of ECM remodeling. NFs with downregulated miR-200 s displayed the traits of activated CAFs, including accelerated migration and invasion. Ectopic expression of miR-200 s in CAFs at least partially restored the phenotypes of NFs. CAF activation may be governed by the targets of miR-200 s, Fli-1 and TCF12, which are responsible for cell development and differentiation; Fli-1 and TCF12 were obviously elevated in CAFs. Furthermore, miR-200 s and their targets influenced collagen contraction by CAFs. The upregulation of fibronectin and lysyl oxidase directly by miR-200 or indirectly through Fli-1 or TCF12 contributed to ECM remodeling, triggering the invasion and metastasis of breast cancer cells both in vitro and vivo. Thus, these data provide important and novel insights into breast CAF activation and ECM remodeling, which trigger tumor cell invasion.It has been well established that a reactive microenvironment induces cancer cells to proliferate, migrate and become invasive. Cancer-associated fibroblasts (CAFs) are thought to be the main players among the cohabitating stromal cell types, and they favor tumor progression. The cancer-promoting ability of CAFs is dependent on their activation; however, this process has not been fully explored.The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans and glycoproteins that exerts biochemical and mechanical effects on cells. An increasing body of evidence suggests that ECM remodeling has an important role in cell morphogenesis,¹ survival,² migration and invasion.³ CAFs can deposit certain ECM components and facilitate the directional migration and invasion of carcinoma cells through mechanotransduction-triggered architectural remodeling of the microenvironment.^{4, 5} However, the mechanism by which activated CAFs stimulate the dysregulation of ECM proteins, thus influencing cancer cell invasion, is not well understood.Previously, our team identified a set of dysregulated miRNAs in breast CAFs using a miRNA microarray, and it was found that the levels of miR-200 family members were noticeably suppressed,⁶ indicating their importance in CAF function. Whether these downregulated miR-200 s in the stroma drive the activated phenotype of CAFs as well as aberrant ECM protein expression to promote cancer cell invasion is an intriguing question.The miR-200 s family can be functionally divided into cluster 1 (miR-200a and miR-141) and cluster 2 (miR-200b and miR-200c) according to their ''seed'' region for binding to mRNA. The effects of the miR-200 s on fibrosis, epithelial cell characteristics, cell differentiation and tumor progression have been discussed. For example, miR-200b is essential for the regulation of renal fibrogenesis⁷ and the intestinal fibrosis of Crohn''s disease.⁸ In aggressive carcinoma cells, the maintenance of EMT,⁹ tumor growth,¹⁰ migration,¹¹ invasion,⁹ anoikis resistance¹² and poor response to chemotherapy¹³ are enhanced by the reduced expression of miR-200 s. Furthermore, miR-200 s are upregulated during mammary differentiation¹⁴ but are downregulated in breast cancer stem cells,¹⁵ and these molecules support the maintenance of pluripotent stem cells.¹⁶ These previous reports indicate that miR-200 s may have a significant role in CAF activation.In the current work, we first determined that miR-200 s were commonly downregulated in breast CAFs, and this result was also demonstrated in normal fibroblasts (NFs) co-cultured with breast cancer cells. miR-200 s induced the conversion of NFs into CAFs by targeting Fli-1 and TCF12. Re-expression of miR-200 s in CAFs attenuated the activation-associated CAF phenotype. In particular, miR-200 s and their targets all contributed to CAF-associated ECM remodeling through two key ECM remodeling proteins, fibronectin (FN) and lysyl oxidase, further fueling cancer cell invasion and metastasis. Therefore, our data provide new information regarding the role of CAF activation and function in the promotion of cancer cell invasion through ECM remodeling and provide a considerable amount of information that will be useful for the development of stromal therapeutic targets.     

MiR-206 regulates neural cells proliferation and apoptosis via Otx2

MiR-206 was involved in a series of cellular activities, such as the growth and development of skeletal muscle and the tumorigenesis. MiR-206 was characterized previously as a differentially expressed gene in sodium arsenite (SA)-induced neural tube defects (NTDs) in chick embryos via miRNA microarray analysis. However, the role of miR-206 in the pathological process of nerve cells remained elusive. In this study we found differential expression of miR-206 in SA-treated chick embryos by Northern blot analysis. Ectopic expression of miR-206 inhibited cell proliferation, and promoted cell apoptosis in U343 and SK-N-SH cell by using MTT, Edu Apollo assay and Flow cytometry analysis. Further investigation revealed that miR-206 can interact with 3'-untranslated region (UTR) of Otx2. MiR-206 mimics down-regulated the endogeneous Otx2 expression, whereas the miR-206 inhibitor obviously up-regulated the expression of Otx2. These findings indicate that overexpression of miR-206 promotes cell apoptosis and low expression of miR-206 inhibits cell apoptosis. Otx2 may play an important role in the process of miR-206-mediated cell apoptosis.     

MicroRNA200a enhances antitumor effects in combination with doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often treated with doxorubicin. MicroRNAs have been shown to have important regulatory roles in cancer and serve as a target in chemoresistance. In this study, we investigated the effects of specific microRNA-200a (miR-200a) on HCC tumor cell growth and effect of doxorubicin-mediated cytotoxicity. Our results show miR-200a is downregulated in human HCC and HCC tumor cell lines. Increasing miR-200a expression inhibited HCC growth and synergized with the antitumor effects of doxorubicin. Inhibiting endogenous miR-200a promoted tumor growth and chemotherapeutic resistance. Increasing miR-200a expression inhibited tumor metabolism (ATP production, mitochondrial respiration, glycolysis), while inhibition of endogenous miR-200a reversed these effects. MiR-200a expression also increased autophagy and synergized with doxorubicin-mediated cytotoxicity. This study identifies a novel role of miR-200a in potentiating doxorubicin-mediated therapeutic effects in HCC.     

Cancer-associated fibroblasts–derived exosomes-mediated transfer of LINC00355 regulates bladder cancer cell proliferation and invasion

The aim of this study was to investigate the regulatory mechanism of cancer-associated fibroblasts (CAFs) exosome in bladder cancer (BC) cell proliferation and invasion. CAFs and normal fibroblasts (NFs) were isolated from tumor tissues and adjacent normal tissues of BC patients, and examined by immunocytochemistry for the expression of fibroblast activation protein alpha (FAP) and α-smooth muscle actin (α-SMA). Exosomes were extracted from CAFs and NFs and observed under a transmission electron microscope, and expression of the exosome markers CD9 and CD63 was confirmed by western blotting. The distribution and intensity of fluorescence were observed by confocal laser microscopy to analyze exosomes uptake by BC cell lines T24 or 5367. BC cell proliferation and invasion were detected by MTT and Transwell assays, respectively. LINC00355 levels in CAFs, NFs, CAFs exosome, NFs exosome, and BC cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that CAFs exosome significantly promoted BC cell proliferation and invasion relative to NFs exosome. LINC00355 expression was significantly elevated in CAFs exosome when compared with that in NFs-exosome. Up-regulated LINC00355 expression was observed both in T24 and 5367 cells co-incubated with CAFs exosome. Exosomes derived from LINC00355 siRNA-transfected CAFs observably repressed BC cell proliferation and invasion when compared with control siRNA-CAFs exosome. In conclusion, CAFs exosome–mediated transfer of LINC00355 regulates BC cell proliferation and invasion. Significance of the study. In this study, our data suggest that the exosomes released from CAFs promote BC cell proliferation and invasion. The mechanism of this effect is, at least in part, related to the increased LINC00355. Regulation of LINC00355 expression in exosomes released from CAFs might be a putative therapeutic strategy against the pathogenesis of BC.     

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.     

Cyclin D1 is a major target of miR-206 in cell differentiation and transformation

miR-206, a member of the so-called myomiR family, is largely acknowledged as a specific, positive regulator of skeletal muscle differentiation. A growing body of evidence also suggests a tumor suppressor function for miR-206, as it is frequently downregulated in various types of cancers. In this study, we show that miR-206 directly targets cyclin D1 and contributes to the regulation of CCND1 gene expression in both myogenic and non-muscle, transformed cells. We demonstrate that miR-206, either exogenous or endogenous, reduces cyclin D1 levels and proliferation rate in C2C12 cells without promoting differentiation, and that miR-206 knockdown in terminally differentiated C2C12 cells leads to cyclin D1 accumulation in myotubes, indicating that miR-206 might be involved in the maintenance of the post-mitotic state. Targeting of cyclin D1 might also account, at least in part, for the tumor-suppressor activity suggested for miR-206 in previous studies. Accordingly, the analysis of neoplastic and matched normal lung tissues reveals that miR-206 downregulation in lung tumors correlates, in most cases, with higher cyclin D1 levels. Moreover, gain-of-function experiments with cancer-derived cell lines and with in vitro transformed cells indicate that miR-206-mediated cyclin D1 repression is directly coupled to growth inhibition. Altogether, our data highlight a novel activity for miR-206 in skeletal muscle differentiation and identify cyclin D1 as a major target that further strengthens the tumor suppressor function proposed for miR-206.     

Chinese Herbal Medicine Suppresses Invasion-Promoting Capacity of Cancer-Associated Fibroblasts in Pancreatic Cancer

Pancreatic cancer remains one of the leading causes of cancer-related deaths, due to aggressive growth, high metastatic rates during the early stage and the lack of an effective therapeutic approach. We previously showed that Qingyihuaji (QYHJ), a seven-herb Chinese medicine formula, exhibited significant anti-cancer effects in pancreatic cancer, associated with modifications in the tumor microenvironment, particularly the inhibition of cancer-associated fibroblast (CAF) activation. In the present study, we generated CAF and paired normal fibroblast (NF) cultures from resected human pancreatic cancer tissues. We observed that CAFs exhibited an enhanced capacity for inducing pancreatic cancer cell migration and invasion compared with NFs, while QYHJ-treated CAFs exhibited decreased migration and invasion-promoting capacities in vitro. The results of further analyses indicated that compared with NFs, CAFs exhibit increased CXCL1, 2 and 8 expression, contributing to the enhanced invasion-promoting capacities of these cells, while QYHJ treatment significantly suppressed CAF proliferation activities and the production of CAF-derived CXCL1, 2 and 8. These in vitro observations were confirmed in mice models of human pancreatic cancer. Taken together, these results suggested that suppressing the tumor-promoting capacity of CAFs through Chinese herbal medicine attenuates pancreatic cancer cell invasion.     

Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal tumors, accounting for almost half a million deaths per year. Cancer-associated fibroblasts (CAFs) are the major constituent of the tumor microenvironment (TME) and dramatically impact ESCC progression. Recent evidence suggests that exosomes derived from CAFs are able to transmit regulating signals and promote ESCC development. In this study, we compared different the component ratios of miRNAs in exosomes secreted by CAFs in tumors and with those from normal fibroblasts (NFs) in precancerous tissues. The mRNA level of hsa-miR-3656 was significantly upregulated in the former exosomes. Subsequently, by comparing tumor cell development in vitro and in vivo, we found that the proliferation, migration and invasion capabilities of ESCC cells were significantly improved when miR-3656 was present. Further target gene analysis confirmed ACAP2 was a target gene regulated by miR-3656 and exhibited a negative regulatory effect on tumor proliferation. Additionally, the downregulation of ACAP2 triggered by exosomal-derived miR-3656 further promotes the activation of the PI3K/AKT and β-catenin signaling pathways and ultimately improves the growth of ESCC cells both in vitro and in xenograft models. These results may represent a potential therapeutic target for ESCC and provide a new basis for clinical treatment plans.