HSP47 Promotes Glioblastoma Stemlike Cell Survival by Modulating

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HSP47 promotes glioblastoma stem-like cell survival by modulating tumor microenvironment extracellular matrix through TGF-# pathway Xiaochun Jiang, Taofeng Zhou, Zhichun Wang, Bin Qi, and Hongping Xia ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00253 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016

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ACS Chemical Neuroscience

HSP47 promotes glioblastoma stem-like cell survival by modulating tumor microenvironment extracellular matrix through TGF-β pathway Xiaochun Jiang1,*, Taofeng Zhou2,*, Zhichun Wang1, Bin Qi3, #, Hongping Xia1,4 # 1

Department of Neurosurgery, Yijishan Hospital, Wannan Medical College, Wuhu

241001, China 2

Department of Neurology, Yijishan Hospital, Wannan Medical College, Wuhu

241001, China 3

Department of Neurosurgery, First Hospital of Jilin University, Changchun, Jilin

130021, P.R. China 4

Department of Pathology, Sir Run Run Hospital & Nanjing Medical University,

Nanjing 211166, China

Running title: HSP47 promotes glioma stemness *These authors contributed equally to this work.

#Correspondence to: Hongping Xia, MD/PhD, Department of Neurosurgery, Yijishan Hospital, Wannan Medical College, Wuhu, Anhui 241001, China, Tel/Fax: +86 553 573 9999, E-mail: [email protected] Bin Qi, MD, Department of Neurosurgery, First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China, Tel/Fax: +86 431 8788 6363, E-mail: [email protected] 1

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Abstract Grade IV glioblastoma multiforme (GBM) is the most malignant form of gliomas. HSP47, encoded by SERPINH1 gene, is a serpin which serves as a human chaperone protein for collagen. We have shown that HSP47 is significantly overexpressed in GBM and associated with tumor grade. However, the role of HSP47 on GBM progression and stem-like property remains unclear. The stable overexpression of HSP47 in primary GBM cells was established by lentivirus infection. The effects of HSP47 overexpression on tumor grow and the effects of blocking TGF-β pathway on tumor regression were investigated by animal study. The expression of HSP47 was examined by real time qRT-PCR and immunohistochemistry. The stem-like property was investigated by sphere formation and CD44 cell population analysis using flow cytometry. We found that overexpression of HSP47 promotes primary glioma cell tumor formation, invasion, angiogenesis and stem-like properties. The overexpression of HSP47 was correlated and promoted extracellular matrix (ECM) related genes through TGF-β pathway in GBM. Blocking TGF-β pathway overcomes HSP47 induced tumorigenesis and stemness. This study demonstrated that HSP47 promotes GBM stem-like cell survival by modulating tumor microenvironment ECM through TGF-β pathway. Blocking TGF-β pathway provides a promising therapeutic potential for HSP47 overexpressed GBM.

Keywords: glioblastoma, HSP47, inhibitor, glioma stem cell, TGF-β, extracellular matrix 2


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INTRODUCTION Gliomas are classified to four malignancy grades, grades I to IV according to the criteria established by the World Health Organization (WHO). Grade IV glioblastoma multiforme (GBM) is the most malignant form of gliomas. GBM has the highly invasive nature that prevents curative tumor resection and resistance to treatment(1). It has been shown that the onset of GBM is driven by a subpopulation of cancer stem cells (CSCs) or Glioma stem cells (GSCs) that display self-renewal and recapitulate tumor heterogeneity(2). GSCs are supported by the tumor microenvironment, which contributes to tumor development and aggressive behavior. The Extracellular matrix (ECM) represents one major part of this “niche”. ECM molecules as well as their complementary receptors contribute to tumor cell behavior and progression(3). Tumor cells exhibit an extensive overexpression of various matrix components to design their own ECM. This matrix in consequence supports the CSCs and tumor cells in their aggressive behavior. ECM plays an important role in processes like migration, angiogenesis, proliferation and differentiation of tumor cells. It is known that the ECM is involved in alterating the diffusion of therapeutic drugs(4). Therefore, targeting tumor ECM constituents might represent a promising therapeutic approach and ECM components might also act as useful diagnosis and prognostic marker molecules. HSP47 is a heat-inducible protein encoded by SERPINH1 gene, which locates at chromosome 11q13.5, one of the most frequently amplified regions in human cancer(5). HSP47 is a member of the serpin superfamily of serine proteinase 3



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inhibitors which serve as a human chaperone protein for collagen. The overexpression of HSP47 has been detected in several tumor tissues including gliomas(6). Our previous study showed that HSP47 is significantly overexpressed in glioma tissues and cell lines and associated with glioma tumor grade. HSP47 regulated by miR-29a to enhance glioma tumor growth and invasion. HSP47 has also been shown to specifically and transiently bind to procollagen, and considered to play important role in collagens maturation and function. Inhibition of HSP47 binding is thought to be an efficient strategy for blocking collagen deposition and ECM remodeling(7). The recent study suggested that HSP47 expression promotes breast cancer progression in part by enhancing deposition of ECM proteins(5). It is well known that the ECM and the surrounding stroma have been shown to play an essential role in glioma progression and stemness(8, 9). However, the function and regulation of HSP47 during glioma progression and GBM stem-like cell survival remains unknown. In this study, we investigated the role and mechanism of HSP47 overexpression on glioma progression and stemness. We found that overexpression of HSP47 promotes primary glioma cell tumor formation and stem-like properties in vivo. Interestingly, we discovered that HSP47 promotes GBM stem-like cell survival by modulating tumor microenvironment extracellular matrix through TGF-β pathway. Blocking TGF-β pathway overcome HSP47 induced tumorigenesis and stemness, which provide a promising therapeutic potential for HSP47 overexpressed GBM.

RESULTS AND DISCUSSION 4


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Overexpression of HSP47 promotes primary glioma cell tumor formation in vivo To further investigate the critical role of HSP47 overexpression in glioma, we overexpressed the HSP47 in two primary GBM cells (GBM1 and GBM2) using lentivirus infection. The overexpression of HSP47 was detected using real time qRT-PCR and an increasing expression of HSP47 (>10 fold) was observed in cells infected with pLenti-HSP47 (Fig. 1A). To determine the in vivo function of HSP47 overexpresssion during glioma progression, the pLenti-control and pLenti-HSP47 infected GBM cells were subcutaneously injected into the flanks of nude mice. Tumor growth was significantly promoted in the HSP47 overexpressed group compared to the control group (Fig. 1B-1D). The tumor tissues were harvest and the IHC staining further validated the overexpression of HSP47 in the tumor tissues of pLenti-HSP47 infected group (Fig. 1E). These data suggested that overexpression of HSP47 promotes primary glioma cell tumor formation in vivo.

The glioma stem-like cell properties was increased by HSP47 overexpression GBM is an aggressive brain tumor whose growth is thought to be driven by stem cell-like cells. To investigate whether HSP47 overexpression induced glioma cell tumor formation was related increasing of glioma stem-like cells, we collected the fresh tumor tissues from both pLenti-control and pLenti-HSP47 infected groups. After dissociation of tissues into single-cell suspensions using the gentleMACS™ dissociator, we detected a consistent increasing of CD44+ cell population in the tumor 5



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cells of pLenti-HSP47 infected groups through flow cytometry analysis (Fig. 2A). CD44 signaling in the glioma perivascular niche has been shown to enhance cancer stem cell phenotypes and promotes aggressive tumor growth(10). The CD44 high expression cell population has also been shown to enrich for GICs and tend to be located in a perivascular niche(11). We further investigated the sphere formation ability of the tumor cells isolated from pLenti-control and pLenti-HSP47 infected groups. The result also showed that overexpression of HSP47 also significantly increased the sphere formation ability of cells (Fig. 2B and 2C), indicated that the glioma stem-like cell properties was increased by HSP47 overexpression.

The overexpression of HSP47 was correlated and promoted ECM related genes in GBM samples Remodeling of the ECM microenvironment is a necessary event for GBM development and progression. To investigate the mechanism of HSP47 induced tumor progression of GBM, we investigated the expression and their correlation to HSP47 of 84 ECM related genes which are important for cell-cell and cell-matrix interactions using the Partek Genomics Suite® software in the GSE4290 dataset with 23 non-tumor samples and 157 tumor samples. Interestingly, we observed that a panel of ECM related genes are highly correlated with the expression of HSP47 (r>0.8 using Pearson correlation analysis) (Fig.3A). Ingenuity Pathway Analysis (IPA) indicated that the function of these genes is mainly related to cell spreading and angiogenesis (Fig.3B and 3C). Using real time qRT-PCR analysis, we further demonstrated that 6


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overexpression of HSP47 significantly increased the expression of ECM related genes, such as CD44, LAMC1m COL4A2, ITGB1, FN1 and MMP9 in the tumors of pLenti-HSP47 infected groups. These data suggested that the overexpression of HSP47 was correlated and promoted ECM related genes in GBM samples.

Overexpression of HSP47 promotes cell invasion and angiogenesis ECM properties have been shown to regulate the migratory response of GBM stem cells (12). The IPA analysis also indicated that the function of HSP47 correlated genes is mainly related to cell spreading and angiogenesis. We further investigate the effects of HSP47 overexpression on the GBM cell invasion and angiogenesis. The in vitro transwell Matrigel invasion assay result indicated that overexpression of HSP47 significantly promotes GBM cell invasion ability (Fig.4A and 4B). To clarify the effect of HSP47 on GBM angiogenesis, we performed in vitro capillary tube formation assays using co-culture of HUVEC cells and isolated GBM cells from both pLenti-control and pLenti-HSP47 infected groups. The result showed that the tube formation ability was significantly increased when co-cultured with isolated GBM cells from pLenti-HSP47 infected groups compared to pLenti-control infected group (Fig.4C and 4D). These data suggested that overexpression of HSP47 promotes cell invasion and angiogenesis.

The HSP47 correlated ECM genes are mainly regulated by TGF-β and CD44 signaling 7



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The IPA analysis also indicated that the HSP47 correlated ECM genes are predicted to be mainly regulated by TGF-β signaling (Fig.5A). We further examined the production of TGF-β in the tumors from both pLenti-control and pLenti-HSP47 infected groups by ELISA. It is interestingly observed more TGF-β production in pLenti-HSP47 infected groups compared to pLenti-control infected group (Fig.5B), suggesting overexpression of HSP47 promotes TGF-β production and activation of TGF-β signaling. Consistently, the expression of HSP47 was validated to be highly correlated with the expression of TGFB1 and TGFB2 in our twenty GBM samples (Fig.5C and 5D. Autocrine TGF-β signaling has been shown to play an essential role in the maintenance of tumorigenicity of GSC(13). Consistently, we also observed the expression of HSP47 was highly correlated with the expression of CD44 in our twenty GBM samples (Fig.5E). Meanwhile, IPA analysis also indicated that the HSP47 correlated ECM genes are also predicted to be regulated by CD44, suggesting the HSP47 correlated ECM genes are mainly regulated by TGF-β and CD44 signaling. . Blocking TGF-β pathway overcome HSP47 induced tumorigenesis and stemness To identify the therapeutic potential on HSP47 overexpressed GBM, we investigated the therapeutic effect of blocking TGF-β pathway using the small molecule inhibitor. Interestingly, the pLenti-HSP47 infected mice tumors were grouped and treated with vehicle or the TGF-β inhibitor (LY2109761, 10mg/kg) for four weeks and sacrificed. The result indicated that TGF-β inhibitor significantly 8


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inhibits tumor growth in the pLenti-HSP47 infected GBM1 and GBM2 tumors (Fig.6A and 6B). We then evaluated the microvessel density by staining the CD31 expression in HSP47 overexpressed tumors and after LY2109761 treatment. The results were showed that overexpression of HSP47 promotes angiogenesis in vivo with more microvessel density with CD31-positive cells. Blocking TGF-beta signing with LY2109761 treatment overcomes the pro-angiogenic effect of HSP47 overexpression in vivo (Fig.6C). At the end point of treatment, the mice were sacrificed and tumor tissues were dissociated into single-cell suspensions. The flow cytometry analysis showed that the CD44+ cell population was significantly decreased in TGF-β inhibitor treated groups (Fig. 6D). These data suggested that blocking TGF-β pathway overcome HSP47 induced tumorigenesis and stemness, which provide promising therapeutic potential for HSP47 overexpressed GBM.

Previously, we have found that HSP47 is significantly overexpressed in glioma tissues and cell lines and associated with glioma tumor grade. We demonstrated that knockdown of HSP47 inhibits glioma cell growth, migration and invasion in vitro and stable knockdown of HSP47 inhibits glioma tumor growth and induces apoptosis in mice models in vivo(6). However, the critical role of HSP47 overexpression in GBM development and progression is still not understanding. In this study, we further found that overexpression of HSP47 promotes primary glioma cell tumor formation and stem-like properties in vivo. Interestingly, we discovered that HSP47 promotes GBM stem-like cell survival by modulating tumor microenvironment ECM through TGF-β 9



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pathway. Abnormal ECM remodeling in GBM has been shown to influence tumor progression, promoting cellular transformation and invasion(14). The interaction between GSCs and specific ECM components is essential to understand how GBM cell-ECM association produces effects on the response of tumor cells to radio- and chemo-therapy. The contributions of ECM interactions to GSC maintenance still remain poorly understood. Laminins have been associated with tumor grade and patient survival in gliomas, particularly laminin-8 is highly expressed in GBM and contributes to tumor invasion and regrowth after therapy (15, 16). Laminin ECM proteins have been found to be localized in the perivascular GBM niche and inform negative patient prognosis. Laminin α2, which is provided by non-GSCs and ECs, has been identified as the critical for GSC maintenance(17). Here, we also observed that the expression of LAMC1, LAMB1 and LAMA2 are highly correlated with the expression of HSP47 and overexpression of HSP47 also significantly increased the expression of LAMC1, suggesting Laminins may play important role on HSP47 mediated effects in GBM. TGF-β signaling is highly active in GBM and elevated TGF-β activity has been associated with poor clinical outcome. TGF-β pathway plays an important role in regulating the behavior of GBM including proliferation, invasion, high angiogenic and immunosuppressive activity. TGF-β signaling has also been connected with GSCs(18). Previous study showed that tissue hypoxia induces the stem cell phenotype through TGF-β-related EMT and contributes to the poor outcome of GBM 10


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patients(19). GSCs are recruited toward endothelial cells and are induced to become pericytes predominantly by TGF-β(20). TGF-β signaling has also been shown to regulate ECM proteins (collagens, fibronection and laminins) and ECM-degrading enzymes, MMPs. Here, we found that the HSP47 correlated ECM genes are mainly regulated by TGF-β and CD44 signaling. CD44 receptor has also been shown to be overexpressed in glioma cells in vitro and found in vivo at the leading edge of glioma at the brain-tumor interface(21). Our study further found that blocking TGF-β pathway overcome HSP47 induced tumorigenesis and stemness. The inhibition of the TGF-β pathway has also been shown to decrease the CD44 (high)/Id1(high) GIC population through the repression of Id1 and Id3 levels, therefore inhibiting the capacity of cells to initiate tumors. High CD44 level also confers poor prognosis in GBM patients (11). In conclusion, this study demonstrated that HSP47 promotes GBM stem-like cell survival by modulating tumor microenvironment extracellular matrix through TGF-β pathway. Blocking TGF-β pathway overcome HSP47 induced tumorigenesis and stemness, which provide a promising therapeutic potential for HSP47 overexpressed GBM.

Material and methods

Cell lines and cell culture The primary GBM cells (GBM1 and GBM2) were established as mentioned 11



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before(22). The glioblastoma tissue samples were collected and proved by the Committees for Ethical Review of Research Involving Human Subjects. The GBM tissue samples were cut into fragments of

HSP47 Promotes Glioblastoma Stemlike Cell Survival by Modulating

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