Hanna E. Rauhala

miR-193b is an epigenetically regulated putative tumor suppressor in prostate cancer.

miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in 6 prostate cancer cell lines and nonmalignant prostate epithelial cells. Thirty‐eight miRNAs showed increased expression in any prostate cancer cell line after 5‐aza‐2′‐deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR‐193b was methylated in 22Rv1 cell line at a CpG island ∼1 kb upstream of the miRNA locus. Expressing miR‐193b in 22Rv1 cells using pre‐miR‐193b oligonucleotides caused a significant growth reduction (p < 0.001) resulting from a decrease of cells in S‐phase of the cell cycle (p < 0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR‐193b‐expressing 22Rv1 cells (p < 0.01). Altogether, our data suggest that miR‐193b is an epigenetically silenced putative tumor suppressor in prostate cancer.

Dual-specificity phosphatase 1 and serum/glucocorticoid-regulated kinase are downregulated in prostate cancer

Inactivation of tumor suppressor genes through deletion, mutation and epigenetic silencing has been shown to occur in cancer. In our study, we combined DNA demethylation and histone deacetylation inhibition treatments with suppression subtraction hybridization (SSH) and cDNA microarrays to identify potentially epigenetically downregulated genes in PC‐3 prostate cancer cell line. We found 11 genes whose expression was upregulated after relieving epigenetic regulation. Expression of 3 genes [dual‐specificity phosphatase 1 (DUSP1), serum/glucocorticoid regulated kinase (SGK) and spermidine/spermine N1‐acetyltransferase (SAT)] was subsequently studied in clinical sample material using real‐time quantitative RT‐PCR and immunohistochemistry. The DUSP1 and SGK mRNA expression was lower in hormone‐refractory prostate carcinomas compared to benign prostate hyperplasia (BPH) or untreated prostate carcinomas. BPH, normal prostate and high‐grade prostate intraepithelial neoplasia (PIN) expressed high levels of DUSP1 and SGK proteins. Ninety‐two percent and 48% of the prostate carcinomas showed almost complete lack of DUSP1 and SGK proteins, respectively, indicating common downregulation of these genes. The genomic bisulphite sequencing did not reveal dense hypermethylation in the promoter regions of either DUSP1 or SGK. In conclusion, the data suggest that downregulation of DUSP1 and SGK is an early event and could be important in the tumorigenesis of prostate cancer.

Clusterin is epigenetically regulated in prostate cancer

Lack of good models has complicated investigations on the mechanisms of prostate cancer. By far, the most commonly used transgenic mouse model of prostate cancer is TRAMP, which, however, has not been fully characterized for genetic and epigenetic aberrations. Here, we screened TRAMP‐derived C2 cell line for the alterations using different microarray approaches, and compared it to human prostate cancer. TRAMP‐C2 had relatively few genomic copy number alterations according to array comparative genomic hybridization (aCGH). However, the gene copy number and expression were significantly correlated (p < 0.001). Screening genes for promoter hypermethylation using demethylation treatment with 5‐aza‐2′‐deoxycytidine and subsequent expression profiling indicated 43 putatively epigenetically silenced genes. Further studies revealed that clusterin is methylated in the TRAMP‐C2 cell line, as well as in the human prostate cancer cell line LNCaP. Its expression was found to be significantly reduced (p < 0.01) in untreated and hormone‐refractory human prostate carcinomas. Together with known function of clusterin, the data suggest an epigenetic component in the regulation of clusterin in prostate cancer.

TCEB1 promotes invasion of prostate cancer cells

Amplification of the long arm of chromosome 8 is one of the most recurrent findings in prostate cancer and it is associated with poor prognosis. Several minimal regions of amplification suggest multiple target genes which are yet to be identified. We have previously shown that TCEB1, EIF3S3, KIAA0196 and RAD21 are amplified and overexpressed in prostate cancer and they are located in the 8q area. In this study, we examined the functional effects of these genes to prostate cancer cell phenotype. We overexpressed and inhibited the genes by lentivirus mediated overexpression and RNA interference, respectively. shRNA mediated TCEB1 silencing decreased significantly cellular invasion of PC‐3 and DU145 cells through Matrigel. TCEB1 silencing reduced the anchorage‐independent growth of PC‐3 cells. Similar effects were not seen with any other genes. When overexpressed in NIH 3T3 cells, TCEB1 and EIF3S3 increased the growth rate of the cells. Transcriptional profiling of TCEB1 silenced PC‐3 cells revealed decrease of genes involved in invasion and metastasis. Finally, we also confirmed here the overexpression of TCEB1 in hormone‐refractory prostate tumors. This study indicates that TCEB1 promotes invasion of prostate cancer cells, is involved in development of hormone‐refractory prostate cancer and is thereby a strong candidate to be one of the target genes for the 8q gain.

Epigenetically altered miR-193b targets cyclin D1 in prostate cancer

Micro‐RNAs (miRNA) are important regulators of gene expression and often differentially expressed in cancer and other diseases. We have previously shown that miR‐193b is hypermethylated in prostate cancer (PC) and suppresses cell growth. It has been suggested that miR‐193b targets cyclin D1 in several malignancies. Here, our aim was to determine if miR‐193b targets cyclin D1 in prostate cancer. Our data show that miR‐193b is commonly methylated in PC samples compared to benign prostate hyperplasia. We found reduced miR‐193b expression (P < 0.05) in stage pT3 tumors compared to pT2 tumors in a cohort of prostatectomy specimens. In 22Rv1 PC cells with low endogenous miR‐193b expression, the overexpression of miR‐193b reduced CCND1 mRNA levels and cyclin D1 protein levels. In addition, the exogenous expression of miR‐193b decreased the phosphorylation level of RB, a target of the cyclin D1‐CDK4/6 pathway. Moreover, according to a reporter assay, miR‐193b targeted the 3’UTR of CCND1 in PC cells and the CCND1 activity was rescued by expressing CCND1 lacking its 3’UTR. Immunohistochemical analysis of cyclin D1 showed that castration‐resistant prostate cancers have significantly (P = 0.0237) higher expression of cyclin D1 compared to hormone‐naïve cases. Furthermore, the PC cell lines 22Rv1 and VCaP, which express low levels of miR‐193b and high levels of CCND1, showed significant growth retardation when treated with a CDK4/6 inhibitor. In contrast, the inhibitor had no effect on the growth of PC‐3 and DU145 cells with high miR‐193b and low CCND1 expression. Taken together, our data demonstrate that miR‐193b targets cyclin D1 in prostate cancer.

KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro

Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7), the newest member of the alpha karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer.

Search for KPNA7 cargo proteins in human cells reveals MVP and ZNF414 as novel regulators of cancer cell growth

Karyopherin alpha 7 (KPNA7) belongs to a family of nuclear import proteins that recognize and bind nuclear localization signals (NLSs) in proteins to be transported to the nucleus. Previously we found that KPNA7 is overexpressed in a subset of pancreatic cancer cell lines and acts as a critical regulator of growth in these cells. This characteristic of KPNA7 is likely to be mediated by its cargo proteins that are still mainly unknown. Here, we used protein affinity chromatography in Hs700T and MIA PaCa-2 pancreatic cancer cell lines and identified 377 putative KPNA7 cargo proteins, most of which were known or predicted to localize to the nucleus. The interaction was confirmed for two of the candidates, MVP and ZNF414, using co-immunoprecipitation, and their transport to the nucleus was hindered by siRNA based KPNA7 silencing. Most importantly, silencing of MVP and ZNF414 resulted in marked reduction in Hs700T cell growth. In conclusion, these data uncover two previously unknown human KPNA7 cargo proteins with distinct roles as novel regulators of pancreatic cancer cell growth, thus deepening our understanding on the contribution of nuclear transport in cancer pathogenesis.

Abstract 76: KPNA7 nuclear import protein - a critical regulator of cancer cell growth

Background. In eukaryotic cells, the nucleus is separated from the cytosol by the nuclear envelope, thus requiring a specific machinery for the transport of macromolecules between the two cellular compartments. The nuclear import cycle is strictly regulated and its malfunction results in incorrect localization of proteins, ultimately leading to variety of diseases including cancer. Karyopherin alpha 7 (KPNA7) is the newest member of the karyopherin alpha family of nuclear importers. KPNA7 is mainly expressed in early embryogenesis and oocytes as well as in some cancer cells. We previously showed that silencing of KPNA7 in pancreatic cancer cell lines with high endogenous expression dramatically reduced cell proliferation and anchorage-independent growth, through a G1 cell cycle arrest and transcriptional induction of p21 expression. Here, we further explored the functional importance of KPNA7 in a large panel of pancreatic and breast cancer cell lines and identified its cargo proteins that may be responsible for the observed phenotypes. Methods. KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers was determined 72 to 96 h after transfection and compared to those in corresponding controls. To isolate KPNA7 cargo proteins, stable tagged KPNA7-overexpressing pancreatic cancer cell lines were established. An affinity-based protein pull-down was performed and the KPNA7-interacting partners were identified with mass spectrometry. Results. The silencing of KPNA7 in six pancreatic and breast cell lines led to decreased proliferation in all cell lines with low to medium expression level, whereas both cancer and normal cell lines with absolutely no KPNA7 expression were not affected, thus confirming the specificity of the phenotype to KPNA7 silencing. The results were also consistent in breast cancer cell lines, implicating a role not restricted to pancreatic cancer. Interestingly, in some cell lines a distinct change from round to lobular nuclear morphology was observed, suggesting that KPNA7 may also participate in the maintenance of nuclear architecture. The protein pull-down followed by mass spectrometry yielded multiple proteins co-purifying with KPNA7. These included several proteins involved in RNA processing and nucleopore proteins. Many of these were also involved in the regulation of cell cycle and p21 pathway, consistent with the phenotype observed in the functional studies. Conclusions. These results implicate KPNA7 as an important regulator of cancer cell growth and suggest that it might also have other functions in the maintenance of cancer cell homeostasis. We were the first to link KPNA7 to a human malignancy and here we expand that notion beyond pancreatic cancer. We have also identified many putative KPNA7 cargos with functions matching the observed cellular phenotypes. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Hanna E. Rauhala, Anne Kallioniemi. KPNA7 nuclear import protein - a critical regulator of cancer cell growth. [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 76.

Abstract 5205: CCND1 is a miR-193b target in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate cancer is the most frequent cancer of men in Western countries and causes second most of the cancer-related deaths. In addition to genetic changes also epigenetic alterations and micro-RNAs (miRNAs) are important in prostate cancer development. Our group has previously shown that miR-193b is hypermethylated in 22Rv1 prostate cancer cell line and that it is putative tumor suppressor in prostate cancer. In melanoma and hepatocellular carcinoma, it has been shown that CCND1, encoding cyclin D1 protein, is among the target genes of miR-193b. The aim of this project was to study if CCND1 is a target of miR-193b in prostate cancer. According to qRT-PCR and microarray analyses, the expression of CCND1 and miR-193b were inversely correlated in prostate cancer cell lines (PC-3, VCaP, 22Rv1, LAPC4, DU145, LNCaP) and in LuCaP xenografts. Using a luciferase reporter gene assay in 22Rv1 cells, we showed that miR-193b targets CCND1 3′UTR. In 22Rv1 cells with low endogenous miR-193b expression, transient pre-miR-193b transfection reduced CCND1 mRNA levels and cyclin D1 and phospho-RB protein levels. By rescue experiment we confirmed that the downregulation of cyclin D1 and phospho-RB proteins is due to miR-193b. When prostate cancer cell lines were treated with cdk4/6 inhibitor, 22Rv1 and VCaP cells with low miR-193b and high CCND1 expression showed significant growth retardation. In contrast, the inhibitor did not have any effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Furthermore, immunohistochemical analysis of 337 specimens showed that cyclin D1 is expressed significantly higher in castration resistant compared to hormone-naive prostate cancers. Therefore, we conclude that CCND1 is one of the miR-193b targets in prostate cancer. Citation Format: Kirsi M. Tuppurainen, Hanna E. Rauhala, Mauro Scaravilli, Robert L. Vessella, Tapio Visakorpi. CCND1 is a miR-193b target in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2014-5205

Abstract LB-272: KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology

Background: Bidirectional nucleocytoplasmic transport unique for eukaryotic cells is a tightly regulated process carried out by specific transport machinery. Its defects result in incorrect localization of proteins that might subsequently lead to diversity of diseases including cancer. Karyopherin alpha 7 (KPNA7) is the newest karyopherin alpha nuclear importer family member, mainly expressed in early embryogenesis and oocytes. However, its expression is reactivated in some cancer cells. We previously showed that KPNA7 promotes the malignant properties of pancreatic cancer cells overexpressing the gene. Here, we identified KPNA7 cargo proteins and further explored the functional consequences of KPNA7 expression for cell proliferation, cell cycle distributions and maintenance of proper nuclear morphology in a panel of pancreatic and breast cancer cell lines with varying KPNA7 expression levels. Methods: To isolate KPNA7 cargo proteins, an affinity-based protein pull-down was performed from stable, tagged KPNA7-overexpressing pancreatic cancer cell lines and the KPNA7 binding partners were identified with mass spectrometry. For functional studies, KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers and cell cycle distributions were determined 72 to 96 h after transfection and compared to those in corresponding controls. To study mitotic spindle assembly and nuclear morphology, immunofluorescent labeling of γ-tubulin and different nuclear envelope proteins was performed 96 h after transfections. Results: The protein pull-down and mass spectrometry yielded multiple proteins co-purifying with KPNA7. Two cargos, zinc finger protein 414 and major vault protein were successfully validated to bind KPNA7 in vitro and shown to be transported into the nucleus by KPNA7. Additionally, these cargos were shown to possess growth regulatory roles in pancreatic cancer cells. The silencing of KPNA7 in six pancreatic and breast cell lines decreased cell proliferation in all cell lines expressing the gene, whereas both cancer and normal cell lines without endogenous KPNA7 expression were not affected. Also, a decrease in the fraction of proliferating S-phase cells was detected. KPNA7 depletion led to aberrant mitotic spindle assembly and abnormal number of centrosomes. Interestingly, a reorganization of nuclear envelope proteins was observed, resulting in distinct change from round to lobular nuclear morphology. Conclusions: The present results indicate KPNA7 as a central regulator of cancer cell growth and cell cycle, and identify KPNA7 cargo proteins participating in this regulation. We also show that KPNA7, probably via its cargos, has roles beyond nuclear transfer. Our results suggest that KPNA7 functions in the regulation of proper mitosis and organization of the mitotic spindle and acts in the maintenance of nuclear envelope structure and nuclear morphology. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Teemu Ihalainen, Hanna E. Rauhala, Anssi Nurminen, Vesa P. Hytonen, Anne Kallioniemi. KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2017-LB-272