Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title: Reduced expression of PROX1 transitions glioblastoma cells into a mesenchymal gene expression subtype Running title: PROX1 regulation of glioblastoma subtype Author list: Kaveh M. Goudarzi1, Jaime A. Espinoza1, Min Guo2, Jiri Bartek1&3, Monica Nistér2, Mikael S. Lindström1, Daniel Hägerstrand2 Author affiliations: 1SciLifeLab, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 21 Stockholm, Sweden. 2Cancer Center Karolinska, Department of Oncology- Pathology, Karolinska Institutet and Karolinska University Hospital at Solna, SE-171 76 Stockholm, Sweden. 3The Danish Cancer Society Research Centre, DK-2100, Copenhagen, Denmark. Corresponding author contact information: email: [email protected] phone: +46 70 569 87 36 address: Department of Oncology-Pathology, Cancer Center Karolinska, CCK R8:05, Karolinska Institutet and Karolinska University Hospital at Solna, SE-171 76 Stockholm, Sweden. Conflict of Interest: The authors have no conflicts of interest to declare. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract The homeodomain transcription factor PROX1 has been linked to several cancer types including gliomas, but its functions remain to be further elucidated. Here we describe a functional role and the prognostic value of PROX1 in glioblastoma. Low expression of PROX1 correlated with poor overall survival and the mesenchymal glioblastoma subtype signature. The latter finding was recapitulated in vitro where suppression or overexpression of PROX1 in glioma cell cultures transitioned cells to a mesenchymal or to a non-mesenchymal glioblastoma gene expression signature, respectively. PROX1 modulation affected proliferation rates that coincided with changes in protein levels of CCNA1 and CCNE1 as well as the cyclin inhibitors CDKN1A, CDKN1B, and CDKN1C. Overexpression of SOX2 increased PROX1 expression, but treatment with a CDK2 inhibitor subsequently decreased PROX1 expression, which was paralleled by decreased SOX2 levels. The THRAP3 protein was a novel binding partner for PROX1, and suppression of THRAP3 increased both transcript and protein levels of PROX1. Together these findings highlight the prognostic value of PROX1 and its role as a regulator of glioblastoma gene expression subtypes, intratumoral heterogeneity, proliferation, and cell cycle control. 2 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Glioblastoma represents the most common and aggressive primary brain tumor type in adults (1). Its intrusive growth and the plastic nature of the tumor make complete surgical resection impossible and the tumor cells prone to evade chemo- and radiation therapy (2). Stem cell regulatory pathways are shown activated in gliomas supporting self-renewal, tumor maintenance and survival under stress (3). Furthermore, glioblastomas are very heterogeneous on an intratumoral level, composed of tumor cells displaying different gene expression signatures constituting the different glioblastoma tumor subtypes (4). Thus, a glioma stem-like phenotype, cell motility and tumor cell heterogeneity are considered significant hurdles to overcome for developing new treatment against these tumors (5,6). Glioblastoma may arise from adult neural stem cells or multipotent neural progenitor cells that persist in proliferative niches in the human central nervous system, or alternatively from differentiated cells of different lineages as for example astrocytes or oligodendrocytes (7). The human glioblastoma transcriptome has been found to resemble normal outer radial glial cells and intermediate progenitors (8). In support of this, it was recently shown that glioblastoma initiation is associated to aberrant reactivation of a normal developmental program in the brain (9). Therefore, a better understanding of developmental pathways and their involvement in glioblastoma is thought to lead to new therapeutic possibilities. PROX1 (Prospero-Related Homeobox 1) is a transcription factor that mediates cell fate decisions of neuroblasts, as reviewed in (10). In several instances PROX1 has been shown to play an active role in cancer. For example, PROX1 suppresses the growth of neuroblastoma (11), whereas it enhances colorectal cancer progression as a - 3 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. catenin/TCF/LEF target gene contributing to a transition from early to a dysplastic stage (12). Prox1 regulates the number of cancer stem cells, by promoting cell proliferation and thereby expanding the cancer stem cell population in intestinal adenomas and colorectal cancer after activation of the Wnt-pathway (13). In human astrocytic brain tumors, the percentage of PROX1+ cells has been shown to increase with tumor grade (14). Furthermore, level of PROX1 predicted survival in grade II gliomas, where a percentage of PROX1+ cells over 10% correlated with worse outcome (15). Moreover, PROX1 has been proposed as a novel pathway-specific prognostic biomarker for high-grade astrocytomas (16). Here we sought further understanding of the role of PROX1 in glioblastoma by analyzing publicly available gene expression data from tumor samples and by performing in vitro experiments. 4 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Material and methods Cell culture Glioblastoma cell lines U-343 MG, U-343 MGa, U-343 MGa Cl2:6, U118MG, U178MG, U373MG, and U1242MG have previously been characterized (17-21) and references therein. The high-grade glioma cultures 4, 11 and 18 were initially characterized based on gene expression and phenotypes (22), and have in other studies also been referred to as U2975, U2982 and U2987 (23), respectively. For unity, these cultures are referred to as U2975(4), U2982(11) and U2987(18) in this study. The cell lines and cultures above were retrieved from authors’ lab stocks (22) and subsequently cultured for less than 3 months. All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) with 10% FBS, 2mM L-glutamine and penicillin-streptomycin at 37°C with 5% CO2, and 5% O2 and >95% humidity. The other glioma cell lines (U87MG, U251MG, M059J and M059K), as well as the osteosarcoma line U2-OS and colon cancer line SW480 were purchased from ATCC and cultured per standard guidelines for less than a month. SOX2 and YFP (control) overexpressing U-343 MG cultures were generated by lentiviral transduction with pLEX-Blast-V5-SOX2 and pLEX- Blast-V5-YFP by methods previously described (24). To confirm the relationship between the U-343 clones they were subjected to STR profiling at NGI-Uppsala, SciLifeLab, Uppsala University, using the AmpFISTR Identifiler PCR Amplification kit (Thermo Fisher) (Table S1). The cultures used in this study were screened for mycoplasma using the MycoAlert™ Mycoplasma Detection Kit (LT07-218, Lonza). Stock solution of CVT-313 (Santa Cruz) was prepared by dissolving powder in DMSO, stored at -20 ℃, and used by dissolving stock solution in cell culture media and incubating with cells at concentration and time indicated. 5 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Lentiviral transduction and transfection of plasmids or siRNA constructs To generate a PROX1 overexpressing cell culture, U-343 MG-PROX1, purified lentivirus (Lentifect™) for PROX1 transduction into U-343 MG was purchased (PROX1 containing virus LPP-F0925-Lv105-100-S and negative control virus LPP-NEG-LV105-100-C, GeneCopoeia), and used according to manufacturer’s instructions. To study PROX1 suppression in U-343 MGa, the cell line U-343 MGa-shPROX1 was generated using lentiviral vectors against PROX1 (TRCN0000232123, Sigma-Aldrich) or unrelated control shRNA targeting GFP (TRCN0000072178, clonetechGfp_438s1c1, previously described (24)). Virus production was performed by calcium-phosphate-mediated co- transfection of HEK 293T cells with packaging plasmids (MISSION Lentiviral Packaging Mix, SHP001, Sigma-Aldrich). 24 hours after transfection the different supernatants were collected two times with 24-hour intervals, filtered and then used to