Shi-Qiong Xu

Mechanism of growth inhibition by MicroRNA 145: The role of the IGF‐I receptor signaling pathway

MicroRNA 145 (miR145) has been proposed as a tumor suppressor. It was previously shown that miR145 targets the 3′ UTR of the insulin receptor substrate‐1 (IRS‐1) and dramatically inhibits the growth of colon cancer cells. miR145 also targets the type 1 insulin‐like growth factor receptor (IGF‐IR). We show here that an IRS‐1 lacking its 3′ UTR is no longer down‐regulated by miR145 and rescues colon cancer cells from miR145‐induced inhibition of growth. An IGF‐IR resistant to miR145 (again by elimination of its 3′ UTR) is not down‐regulated by miR145 but fails to rescue colon cancer cells from growth inhibition. These and other results, taken together, indicate that down‐regulation of IRS‐1 plays a significant role in the tumor suppressor activity of miR145. J. Cell. Physiol. 220: 485–491, 2009.

The role of mGrb10alpha in insulin-like growth factor I-mediated growth.

Several isoforms of Grb10 are known to interact with either the insulin receptor or the insulin-like growth factor I (IGF-I) receptor, or both. Inasmuch as the data in the literature on the function of Grb10 are not always concordant, we have investigated the role of one of these isoforms, mGrb10α, in cell proliferation. For this purpose, a plasmid expressing mGrb10α was stably transfected into p6 cells and other mouse embryo fibroblast cell lines. An overexpressed mGrb10α inhibits IGF-I-mediated growth, causes a delay in the S and G2 phases of the cell cycle, and partially reverses the transformed phenotype. In contrast, it has no effect on insulin stimulation of cell proliferation. These studies indicate that this isoform of Grb10 has an inhibitory effect on IGF-I signaling of cell proliferation.

Decorin Antagonizes IGF Receptor I (IGF-IR) Function by Interfering with IGF-IR Activity and Attenuating Downstream Signaling

We have recently discovered that the insulin-like growth factor receptor I (IGF-IR) is up-regulated in human invasive bladder cancer and promotes migration and invasion of transformed urothelial cells. The proteoglycan decorin, a key component of the tumor stroma, can positively regulate the IGF-IR system in normal cells. However, there are no available data on the role of decorin in modulating IGF-IR activity in transformed cells or in tumor models. Here we show that the expression of decorin inversely correlated with IGF-IR expression in low and high grade bladder cancers (n = 20 each). Decorin bound with high affinity IGF-IR and IGF-I at distinct sites and negatively regulated IGF-IR activity in urothelial cancer cells. Nanomolar concentrations of decorin promoted down-regulation of IRS-1, one of the critical proteins of the IGF-IR pathway, and attenuated IGF-I-dependent activation of Akt and MAPK. This led to decorin-evoked inhibition of migration and invasion upon IGF-I stimulation. Notably, decorin did not cause down-regulation of the IGF-IR in bladder, breast, and squamous carcinoma cells. This indicates that decorin action on the IGF-IR differs from its known activity on other receptor tyrosine kinases such as the EGF receptor and Met. Our results provide a novel mechanism for decorin in negatively modulating both IGF-I and its receptor. Thus, decorin loss may contribute to increased IGF-IR activity in the progression of bladder cancer and perhaps other forms of cancer where IGF-IR plays a role.

The insulin-like growth factor receptor I promotes motility and invasion of bladder cancer cells through Akt- and mitogen-activated protein kinase-dependent activation of paxillin.

The insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. Aberrant IGF-IR signaling is implicated in several types of tumors, including carcinomas of the lung, breast, prostate, pancreas, liver, and colon. However, the contribution of the IGF-IR to the development of the transformed phenotype in urothelial cells has not been clearly established. In this study we demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues compared with nonmalignant controls. We have investigated the role of the IGF-IR in bladder cancer by using urothelial carcinoma-derived 5637 and T24 cells. Although activation of the IGF-IR did not appreciably affect their growth, it did promote migration and stimulate in vitro wound closure and invasion. These effects required the activation of the Akt and Mitogen-activated protein kinase (MAPK) pathways as well as IGF-I-induced Akt- and MAPK-dependent phosphorylation of paxillin, which relocated at dynamic focal adhesions and was necessary for promoting motility in bladder cancer cells. Our results provide the first evidence for a role of the IGF-IR in motility and invasion of bladder cancer cells and support the hypothesis that the IGF-IR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia. Thus, the IGF-IR may also serve as a novel biomarker for bladder cancer.

Co-operation of Simian virus 40 T antigen and insulin receptor substrate-1 in protection from apoptosis induced by Interleukin-3 withdrawal

32D cells are interleukin-3 (IL-3) dependent murine hemopoietic cells, that undergo apoptosis after IL-3 withdrawal. An overexpressed insulin-like growth factor I receptor (IGF-IR) protects these cells from apoptosis induced by IL-3 withdrawal. When 32D cells are stably transfected with plasmids expressing either IRS-1 (a major substrate of the IGF-IR) or the Simian virus 40 large T antigen, singly, they still undergo apoptosis after IL-3 withdrawal, although IRS-1 offers partial protection. The cells, however, are fully protected when they are stably transfected with both IRS-1 and SV40 T antigen. Protection from apoptosis in these cells is characterized by the stabilization of the Stat1 and Stat5 protein levels, whose synthesis is inhibited when IL-3 is withdrawn.

Proepithelin Regulates Prostate Cancer Cell Biology by Promoting Cell Growth, Migration, and Anchorage-Independent Growth

The growth factor proepithelin has recently emerged as an important regulator of transformation in several physiological and pathological systems. In this study, we determined the biological roles of proepithelin in prostate cancer cells using purified human recombinant proepithelin as well as proepithelin-depletion strategies. Proepithelin promoted the migration of androgen-dependent and -independent human prostate cancer cells; androgen-independent DU145 cells were the more responsive. In these cells, proepithelin additionally stimulated wound closure, invasion, and promotion of cell growth in vitro. These effects required the activation of both the Akt and mitogen-activated protein kinase pathways. We have analyzed proepithelin expression levels in different available prostate cancer microarray studies using the Oncomine database and found a statistically significant increase in proepithelin mRNA expression levels in prostate cancers compared with nonneoplastic controls. Notably, depletion of endogenous proepithelin by siRNA and antisense strategies impaired the ability of DU145 cells to grow and migrate after serum withdrawal and inhibited anchorage-independent growth. Our results provide the first evidence for a role of proepithelin in stimulating the migration, invasion, proliferation, and anchorage-independent growth of prostate cancer cells. This study supports the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and initial progression of prostate cancer. Furthermore, proepithelin may prove to be a useful clinical marker for the diagnosis of prostate tumors.

Proepithelin is an autocrine growth factor for bladder cancer.

The growth factor proepithelin functions as an important regulator of proliferation and motility. Proepithelin is overexpressed in a great variety of cancer cell lines and clinical specimens of breast, ovarian and renal cancer, as well as glioblastomas. Using recombinant proepithelin on 5637 transitional cell carcinoma-derived cells, we have shown previously that proepithelin plays a critical role in bladder cancer by promoting motility of bladder cancer cells. In this study, we used the ONCOMINE database and gene microarray analysis tool to analyze proepithelin expression in several bladder cancer microarray studies. We found a statistically significant increase in proepithelin messenger RNA expression in bladder cancers vis-à-vis non-neoplastic tissues, and this was associated with pathologic and prognostic parameters. Targeted downregulation of proepithelin in T24 transitional carcinoma cells with small hairpin RNA inhibited both Akt and mitogen-activated protein kinase pathways, severely reduced the ability of T24 cells to proliferate in the absence of serum and inhibited migration, invasion and wound healing. In support of these in vitro results, we discovered that proepithelin expression was significantly upregulated in invasive bladder cancer tissues compared with normal urothelium. In addition, proepithelin was secreted in the urine, where it was detectable by immunoblotting and enzyme-linked immunosorbent assay. Collectively, these results support the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and progression of bladder cancer and suggest that proepithelin may prove a novel biomarker for the diagnosis and prognosis of bladder neoplasms.

Decorin differentially modulates the activity of insulin receptor isoform A ligands

The proteoglycan decorin, a key component of the tumor stroma, regulates the action of several tyrosine-kinase receptors, including the EGFR, Met and the IGF-IR. Notably, the action of decorin in regulating the IGF-I system differs between normal and transformed cells. In normal cells, decorin binds with high affinity to both the natural ligand IGF-I and the IGF-I receptor (IGF-IR) and positively regulates IGF-IR activation and downstream signaling. In contrast, in transformed cells, decorin negatively regulates ligand-induced IGF-IR activation, downstream signaling and IGF-IR-dependent biological responses. Whether decorin may bind another member of the IGF-I system, the insulin receptor A isoform (IR-A) and its cognate ligands, insulin, IGF-II and proinsulin, have not been established. Here we show that decorin bound with high affinity insulin and IGF-II and, to a lesser extent, proinsulin and IR-A. We utilized as a cell model system mouse embryonic fibroblasts homozygous for a targeted disruption of the Igf1r gene (designated R(-) cells) which were stably transfected with a human construct harboring the IR-A isoform of the receptor. Using these R(-)/IR-A cells, we demonstrate that decorin did not affect ligand-induced phosphorylation of the IR-A but enhanced IR-A downregulation after prolonged IGF-II stimulation without affecting insulin and proinsulin-dependent effects on IR-A stability. In addition, decorin significantly inhibited IGF-II-mediated activation of the Akt pathways, without affecting insulin and proinsulin-dependent signaling. Notably, decorin significantly inhibited IGF-II-mediated cell proliferation of R(-)/IR-A cells but affected neither insulin- nor proinsulin-dependent mitogenesis. Collectively, these results suggest that decorin differentially regulates the action of IR-A ligands. Decorin preferentially inhibits IGF-II-mediated biological responses but does not affect insulin- or proinsulin-dependent signaling. Thus, decorin loss may contribute to tumor initiation and progression in malignant neoplasms which depend on an IGF-II/IR-A autocrine loop.

Proline-rich tyrosine kinase 2 (Pyk2) regulates IGF-I-induced cell motility and invasion of urothelial carcinoma cells

The insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. We have recently demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues and promotes motility and invasion of urothelial carcinoma cells. These effects require IGF-I-induced Akt- and MAPK-dependent activation of paxillin. The latter co-localizes with focal adhesion kinases (FAK) at dynamic focal adhesions and is critical for promoting motility of urothelial cancer cells. FAK and its homolog Proline-rich tyrosine kinase 2 (Pyk2) modulate paxillin activation; however, their role in regulating IGF-IR-dependent signaling and motility in bladder cancer has not been established. In this study we demonstrate that FAK was not required for IGF-IR-dependent signaling and motility of invasive urothelial carcinoma cells. On the contrary, Pyk2, which was strongly activated by IGF-I, was critical for IGF-IR-dependent motility and invasion and regulated IGF-I-dependent activation of the Akt and MAPK pathways. Using immunofluorescence and AQUA analysis we further discovered that Pyk2 was overexpressed in bladder cancer tissues as compared to normal tissue controls. Significantly, in urothelial carcinoma tissues there was increased Pyk2 localization in the nuclei as compared to normal tissue controls. These results provide the first evidence of a specific Pyk2 activity in regulating IGF-IR-dependent motility and invasion of bladder cancer cells suggesting that Pyk2 and the IGF-IR may play a critical role in the invasive phenotype in urothelial neoplasia. In addition, Pyk2 and the IGF-IR may serve as novel biomarkers with diagnostic and prognostic significance in bladder cancer.

Abstract 698: Progranulin targeting in urothelial cancer cells inhibits motility, tumor growth in vitro and in vivo and sensitizes cells to cisplatin

Introduction and Objective: Bladder cancer is a major public health problem and affects more than 74,000 Americans with more than 16,000 estimated deaths in 2015. The majority of deaths are due to metastatic spread, commonly to the lungs. Understanding the molecular mechanisms regulating bladder tumor cell invasion and progression toward metastases is essential for developing better therapies to treat bladder cancer patients. The growth factor progranulin has emerged in recent years as an important regulator of transformation in several cancer models. We have previously established a critical role for progranulin in bladder cancer as in fact progranulin acts as an autocrine growth factor and promote motility and invasion of invasive urothelial cancer cells. In addition, progranulin is upregulated in high grade bladder cancer tissues compared to normal tissue controls suggesting that progranulin might work as a novel biomarker with predictive value for bladder cancer progression. However, whether progranulin is important for anchorage-independent growth and in vivo tumor formation of urothelial cancer cells has not been previously established. Methods: Progranulin depletion was achieved by stably transfecting tumorigenic T24T, UMUC3 urothelial cancer cells with a plasmid expressing an anti-progranulin shRNA. Progranulin-depleted and control UMUC-3 and T24T cells were tested for motility, invasion and anchorage-independent growth by soft-agar assays. Tumor formation in vivo was assessed in various UMUC-3-derived cell lines by xenograft and orthotopic models. Sensitivity to cisplatin was assessed by cell survival curves. Progranulin expression levels in a bladder tissue microarray were analyzed by HIC. Results: Progranulin-depleted T24T and UMUC-3 cells were significantly inhibited in their ability to migrate, close a wound and invade through Matrigel compared to control cells in both serum-deprived and 1% serum media. In addition, progranulin targeting strongly reduced the ability of T24T and UMUC-3 cells to grow in anchorage-independency and form colonies in soft-agar. Significantly progranulin-depleted UMUC-3 cells were severely inhibited in tumor formation in vivo as assessed by both xenograft and orthotopic models in immunocompromised mice. Importantly, progranulin depletion sensitized UMUC-3 cells to cisplatin. Finally, progranulin levels correlated with tumor progression in bladder cancer tissues. Conclusions: Our data are translationally relevant as indicate that progranulin exerts an essential functional role in the regulation of bladder cancer progression. Thus, progranulin may constitute a novel target for therapeutic intervention in bladder tumors. In addition, progranulin may serve as novel biomarker for diagnosis and prognosis of bladder cancer. Citation Format: Simone Buraschi, Shi-Qiong Xu, Manuela Stefanello, Igor Moskalev, Alaide Morcavallo, Marco Genua, Ryuta Tanimoto, Thomas Neill, Stephen C. Peiper, Leonard G. Gomella, Antonino Belfiore, Peter C. Black, Renato V. Iozzo, Andrea Morrione. Progranulin targeting in urothelial cancer cells inhibits motility, tumor growth in vitro and in vivo and sensitizes cells to cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 698.