Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell growth and metastasis. We investigated the roles of flow-induced shear stress in modulating cell cycle distribution in four tumor cell lines and the underlying mechanisms. In all four cell lines, incubation under static conditions for 24 or 48 h led to G0/G1 arrest; in contrast, shear stress (12 dynes/cm 2 ) induced G2/M arrest. The molecular basis of the shear effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 osteosarcoma cells. Shear stress induced increased expressions of cyclin B1 and p21 CIP1 and decreased expressions of cyclins A, D1, and E, cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27 KIP1 as well as a decrease in Cdk1 activity. Using specific antibodies and small interfering RNA, we found that the shear-induced G2/M arrest and corresponding changes in G2/M regulatory protein expression and activity were mediated by ␣v␤3 and ␤1 integrins through bone morphogenetic protein receptor type IA-specific Smad1 and Smad5. Shear stress also down-regulated runt-related transcription factor 2 (Runx2) binding activity and osteocalcin and alkaline phosphatase expressions in MG63 cells; these responses were mediated by ␣v␤3 and ␤1 integrins through Smad5. Our findings provide insights into the mechanism by which shear stress induces G2/M arrest in tumor cells and inhibits cell differentiation and demonstrate the importance of mechanical microenvironment in modulating molecular signaling, gene expression, cell cycle, and functions in tumor cells.M echanical microenvironment plays important roles in modulating tissue development, maintenance, and remodeling and in cellular responses and functions (1). Interstitial fluid flow in and around tissue affects the mechanical microenvironment, including the shear stress and pressure force acting on the cell surface and the tethering force acting on cell-matrix connections (2). These interstitial flow-induced forces can modulate tumor metastasis and invasion as well as anticancer drug delivery (3).Although the influence of interstitial fluid flow on tumor pathobiology and drug delivery has been studied, the effect of the flow-induced shear force on tumor cells has not been much explored. Compressive forces have been shown to inhibit tumor cell growth (4) and up-regulate adhesion molecules (5). A recent study reported that tumor cell proliferation is affected by intratumoral pressure and that activation of mitogen-activated protein kinases and nuclear antigen Ki-67 is involved in this mechanical modulation (6). Although these results show that mechanical forces can modulate tumor cell responses, the detailed mechanisms by which mechanical stimuli are transduced into cellular signaling to regulate tumor cell gene expression and functions remain unclear.Integrins have been implicated as mechanosensors in many types of cells seeded on extracellular matrix (ECM) (7), but their role in modulating mechanical responses of...

Recent studies indicated that the estrogen receptor beta (ERβ) could affect the progression of prostate and bladder tumors, however, its roles in the renal cell carcinoma (RCC), remain to be elucidated. Here, we provide clinical evidence that ERβ expression is correlated in a negative manner with the overall survival/disease-free survival in RCC patients. Mechanism dissection revealed that targeting ERβ with ERβ-shRNA and stimulating the transactivation of ERβ with 17β-estradiol or environmental endocrine disrupting chemicals, all resulted in altering the lncRNA HOTAIR expression. The ERβ-modulated HOTAIR is able to function via antagonizing several microRNAs, including miR-138, miR-200c, miR-204, or miR-217 to impact various oncogenes, including ADAM9, CCND2, EZH2, VEGFA, VIM, ZEB1, and ZEB2, to promote RCC proliferation and invasion. Together, the identification of the ERβ-HOTAIR axis may provide us new biomarkers and/or therapeutic targets to better suppress RCC progression in the future.

Early clinical studies suggested that infiltrating mast cells could be associated with a poor outcome in bladder cancer (BCa) patients. The mechanisms of how mast cells influence the BCa progression, however, are unclear. Using the human clinical BCa sample survey and in vitro co-culture systems, we found BCa cells could recruit more mast cells than the surrounding non-malignant urothelial cells. The consequences of this better recruitment of mast cells toward BCa cells could then enhance BCa cell invasion. Mechanism dissection revealed that the enhanced BCa cell invasion could function via up-regulation of the estrogen receptor beta (ERβ) in both mast cells and BCa cells, which resulted in the increased CCL2/CCR2/EMT/MMP9 signals. Using the pre-clinical mouse BCa model, we further validated the mast cell-promoted BCa invasion. Interruption of the newly identified ERβ/CCL2/CCR2/EMT/MMP9 pathway via either ERβ-siRNA, ERβ antagonist PHTPP, or CCR2 antagonist can effectively reverse the mast cell-enhanced BCa cells invasion. Together, our finding could lead to the development of an alternative new therapeutic approach to better treat BCa metastasis.

BACKGROUND Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. METHODS To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. RESULTS The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. CONCLUSIONS Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer.

BackgroundCancer associated fibroblasts (CAF) play important roles in tumor growth that involves inflammation and epithelial cell differentiation. Early studies suggested that estrogen receptor alpha (ERα) was expressed in stromal cells in normal prostates and prostate cancer (PCa), but the detailed functions of stromal ERα in the PCa remain to be further elucidated.MethodsMigration and invasion assays demonstrated the presence of high levels of ERα in CAF cells (CAF.ERα(+)) suppressed PCa invasion via influencing the infiltration of tumor associated macrophages. ERα decreased CAF CCL5 secretion via suppressing the CCL5 promoter activity was examined by luciferase assay. ERα decreased CCL5 and IL-6 expression in conditioned media that was collected from CAF cell only or CAF cell co-cultured with macrophages as measured by ELISA assay.ResultsBoth in vitro and in vivo studies demonstrated CAF.ERα(+) led to a reduced macrophage migration toward PCa via inhibiting CAF cells secreted chemokine CCL5. This CAF.ERα(+) suppressed macrophage infiltration affected the neighboring PCa cells invasion and the reduced invasiveness of PCa cells are at least partly due to reduced IL6 expression in the macrophages and CAF.ConclusionOur data suggest that CAF ERα could be applied as a prognostic marker to predict cancer progression, and targeting CCL5 and IL6 may be applied as an alternative therapeutic approach to reduce M2 type macrophages and PCa invasion in PCa patients with low or little ERα expression in CAF cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0488-9) contains supplementary material, which is available to authorized users.

Neutrophils make up a significant portion of the infiltrated immune cells found in the tumor microenvironment. Here we found more infiltrated neutrophils in human renal cell carcinoma (RCC) lesions than adjacent benign areas. In vitro RCC studies showed that neutrophils (HL-60N cells) infiltrated toward RCC cells and subsequently enhanced RCC cell migration and invasion. Co-culture of RCC cells with HL-60N cells up-regulated ERβ, VEGFa and HIF2α mRNA levels. ERβ signals increased RCC cell migration via induction of the VEGFa/HIF2α pathway. Treatment of HIF inhibitor or rapamycin, or knockdown of ERβ in RCC cells reversed HL-60N-promoted RCC migration. In vivo data using orthotopically xenografted RCC mouse model confirmed that infiltrated neutrophils promoted RCC migration via modulating the expressions of ERβ, VEGFa and HIF2α signal pathways. Together, our studies revealed that neutrophils are favorably recruited to the RCC cells to promote the RCC migration and invasion. Targeting the infiltrating RCC tumor microenvironment with anti-estrogen or rapamycin may be a potential therapy to suppress RCC progression.

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BackgroundGenetically engineered mouse models are essential to the investigation of the molecular mechanisms underlying human prostate pathology and the effects of therapy on the diseased prostate. Serial in vivo volumetric imaging expands the scope and accuracy of experimental investigations of models of normal prostate physiology, benign prostatic hyperplasia and prostate cancer, which are otherwise limited by the anatomy of the mouse prostate. Moreover, accurate imaging of hyperplastic and tumorigenic prostates is now recognized as essential to rigorous pre-clinical trials of new therapies. Bioluminescent imaging has been widely used to determine prostate tumor size, but is semi-quantitative at best. Magnetic resonance imaging can determine prostate volume very accurately, but is expensive and has low throughput. We therefore sought to develop and implement a high throughput, low cost, and accurate serial imaging protocol for the mouse prostate.MethodsWe developed a high frequency ultrasound imaging technique employing 3D reconstruction that allows rapid and precise assessment of mouse prostate volume. Wild-type mouse prostates were examined (n = 4) for reproducible baseline imaging, and treatment effects on volume were compared, and blinded data analyzed for intra- and inter-operator assessments of reproducibility by correlation and for Bland-Altman analysis. Examples of benign prostatic hyperplasia mouse model prostate (n = 2) and mouse prostate implantation of orthotopic human prostate cancer tumor and its growth (n = 6) are also demonstrated.ResultsSerial measurement volume of the mouse prostate revealed that high frequency ultrasound was very precise. Following endocrine manipulation, regression and regrowth of the prostate could be monitored with very low intra- and interobserver variability. This technique was also valuable to monitor the development of prostate growth in a model of benign prostatic hyperplasia. Additionally, we demonstrate accurate ultrasound image-guided implantation of orthotopic tumor xenografts and monitoring of subsequent tumor growth from ~10 to ~750 mm3 volume.DiscussionHigh frequency ultrasound imaging allows precise determination of normal, neoplastic and hyperplastic mouse prostate. Low cost and small image size allows incorporation of this imaging modality inside clean animal facilities, and thereby imaging of immunocompromised models. 3D reconstruction for volume determination is easily mastered, and both small and large relative changes in volume are accurately visualized. Ultrasound imaging does not rely on penetration of exogenous imaging agents, and so may therefore better measure poorly vascularized or necrotic diseased tissue, relative to bioluminescent imaging (IVIS).ConclusionsOur method is precise and reproducible with very low inter- and intra-observer variability. Because it is non-invasive, mouse models of prostatic disease states can be imaged serially, reducing inter-animal variability, and enhancing the power to detect small volume changes following therapeut...