The overexpression of P21-activated kinase 5 (PAK5) promotes paclitaxel-chemoresistance of epithelial ovarian cancer

Protein kinases are important regulators in biologic processes. Aberrant expression of protein kinases often causes diseases including cancer. In the present study, we found that the serine-arginine protein kinase 1 (SRPK1) might be involved in hepatocellular carcinoma (HCC) proliferation from a kinome screen using a loss-of-function approach. In clinical samples, SRPK1 was frequently up-regulated in HCCs as compared with adjacent non-tumor tissues at both mRNA and protein levels. Functional studies indicated that overexpression of wild-type SRPK1 promoted HCC cell proliferation, while forced expression of the kinase-dead mutant of SRPK1 or RNA interference against SRPK1 suppressed cell growth and malignancy as measured in soft agar assay. The kinase-dead mutant of SRPK1 also inhibited subcutaneous xenografts’ growth of HCC cells in nude mice. Furthermore, western bolt analysis showed overexpression of wild-type SRPK1 enhanced Akt phosphorylation and knockdown of SRPK1 by RNA interference attenuated Akt phosphorylation induced by epidermal growth factor. Meanwhile, overexpression of wild-type SRPK1 also induced a concurrent increase in the total tyrosine phosphorylation of phosphotidylinositol-3 kinase p110α subunit, indicating a functional link between SRPK1 and PI3K/Akt signaling. Our findings suggest that SRPK1 plays an oncogenic role and could be a potential therapeutic target in HCC.     

RETRACTED ARTICLE: The Critical Role of SRPK1 in EMT of Human Glioblastoma in the Spinal Cord

Up to now, the serine-arginine protein kinase 1 (SRPK1) has been suggested as an important signal mediator, which is implicated in the development of cancers. Unfortunately, some molecular pathways in SRPK1-mediated epithelial-mesenchymal transition (EMT) in human spinal glioblastoma have been not elucidated. In this work, we detected the expression of SRPK1 in human spinal glioblastoma tissues and GBM cell lines and analyzed the relevant molecular proteins using in vitro experiments, including RT-PCR, gene silencing, and Western blot. In this study, RT-PCR and Western blot revealed that the expression of SRPK1 mRNA and protein became higher in all six spinal glioblastoma specimens; however, its expression was low in matched normal specimens. We also demonstrated SRPK1 expression facilitated the proliferation of U87 and U251 cells and inhibited the apoptosis in U87 and U251 cells. Also, SRPK1 promoted the expression of EMT-regulating markers, involving N-cadherin, Snail, and MMP9 and decreased the expression of mesenchymal marker E-cadherin. Moreover, knockdown of SRPK1 significantly inhibited the expression levels of p-Akt rather than t-Akt. In conclusion, knockdown of SRPK1 inhibited glioblastoma cell proliferation, invasion, and EMT process via suppressing p-Akt signaling pathway. This study also lays a new foundation for the clinically biological treatment.     

The E1circumflexE4 protein of human papillomavirus interacts with the serine-arginine-specific protein kinase SRPK1

Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1circumflexE4 protein during the productive phase of infection. However, the precise mechanisms of how E1circumflexE4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1circumflexE4. We map critical residues within an arginine-rich domain of HPV1 E1circumflexE4, and in a region known to facilitate E1circumflexE4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1circumflexE4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1circumflexE4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1circumflexE4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1circumflexE4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1circumflexE4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.     

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.     

SRPK1 facilitates tumor cell growth via modulating the small nucleolar RNA expression in gastric cancer

Serine-arginine protein kinase 1 (SRPK1) is the main regulator in alternative splicing by phosphorylating splicing factors rich in serine/arginine repeats. Its overexpression has been found in multiple cancer types and contributes to cancer development. Here we report the role of SRPK1 and underlying mechanism in gastric cancer (GC) cell growth. We found that SRPK1 was frequently upregulated in GC samples compared with their adjacent corresponding normal tissues by immunohistochemistry and western blot analysis. Knockdown of SRPK1 in GC cells suppressed cell growth in cell viability assays, colony formation assays and nude mice xenograft model, whereas overexpression of SRPK1 promotes opposite phenotypes in these assays. By a complementary DNA microarray analysis, we found that SRPK1 knockdown had significant inhibitory effects on a majority of small nucleolar RNAs expression. Among them, snoRA42, snoRA74A, and snoRD10 were selected for further functional experiments. Cell growth curves on a plate and in soft agar indicated that the three snoRNAs play potential oncogenic function in GC. In addition, SRPK1 could co-immunoprecipitated with NCL, a nucleolar phosphoprotein involved in the synthesis and maturation of ribosomes. These results suggested that SRPK1 contributes to GC development by a new possible mechanism involving snoRNAs mediated signaling.     

Atypical Protein Kinase C Zeta: Potential Player in Cell Survival and Cell Migration of Ovarian Cancer

Ovarian cancer is one of the most aggressive gynaecological cancers, thus understanding the different biological pathways involved in ovarian cancer progression is important in identifying potential therapeutic targets for the disease. The aim of this study was to investigate the potential roles of Protein Kinase C Zeta (PRKCZ) in ovarian cancer. The atypical protein kinase C isoform, PRKCZ, is involved in the control of various signalling processes including cell proliferation, cell survival, and cell motility, all of which are important for cancer development and progression. Herein, we observe a significant increase in cell survival upon PRKCZ over-expression in SKOV3 ovarian cancer cells; additionally, when the cells are treated with small interference RNA (siRNA) targeting PRKCZ, the motility of SKOV3 cells decreased. Furthermore, we demonstrate that over-expression of PRKCZ results in gene and/or protein expression alterations of insulin-like growth factor 1 receptor (IGF1R) and integrin beta 3 (ITGB3) in SKOV3 and OVCAR3 cells. Collectively, our study describes PRKCZ as a potential regulatory component of the IGF1R and ITGB3 pathways and suggests that it may play critical roles in ovarian tumourigenesis.     

Osteopontin-c splicing isoform contributes to ovarian cancer progression

Ovarian carcinoma is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is one of the proteins overexpressed in ovarian cancer and is involved in tumorigenesis and metastasis. Alternative splicing of OPN leads to 3 isoforms, OPNa, OPNb, and OPNc. However, the expression pattern and the roles of each of these isoforms have not been previously characterized in ovarian cancer. Herein, we have evaluated the expression profiling of OPN isoforms in ovarian tumor and nontumor samples and their putative roles in ovarian cancer biology using in vitro and in vivo functional assays. OPNa and OPNb were expressed both in tumor and nontumor ovarian samples, whereas OPNc was specifically expressed in ovarian tumor samples. The isoform OPNc significantly activated OvCar-3 cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in vivo. Additionally, we have also shown that some of the OPNc-dependent protumorigenic roles are mediated by PI3K/Akt signaling pathway. OPNc stimulated immortalized ovarian epithelial IOSE cell proliferation, indicating a role for this isoform in ovarian cancer tumorigenesis. Functional assays using OPNc conditioned medium and an anti-OPNc antibody have shown that most cellular effects observed herein were promoted by the secreted OPNc. According to our data, OPNc-specific expression in ovarian tumor samples and its role on favoring different aspects of ovarian cancer progression suggest that secreted OPNc contributes to the physiopathology of ovarian cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for ovarian cancer that selectively down regulate OPNc, altering its properties favoring ovarian tumor progression.     

Arf6, RalA and BIRC5 protein expression in non small cell lung cancer

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.     

Implications of Proprotein Convertases in Ovarian Cancer Cell Proliferation and Tumor Progression: Insights for PACE4 as a Therapeutic Target

Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target.     

FBXO16-mediated hnRNPL ubiquitination and degradation plays a tumor suppressor role in ovarian cancer

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a type of RNA binding protein that highly expressed in a variety of tumors and plays a vital role in tumor progression. However, its post-translational regulation through ubiquitin-mediated proteolysis and the cellular mechanism responsible for its proteasomal degradation remains unclear. F-box proteins (FBPs) function as the substrate recognition subunits of SCF ubiquitin ligase complexes and directly bind to substrates. The aberrant expression or mutation of FBPs will lead to the accumulation of its substrate proteins that often involved in tumorigenesis. Here we discover FBXO16, an E3 ubiquitin ligase, to be a tumor suppressor in ovarian cancer, and patients with the relatively high expression level of FBXO16 have a better prognosis. Silencing or depleting FBXO16 significantly enhanced ovarian cancer cell proliferation, clonogenic survival, and cell invasion by activating multiple oncogenic pathways. This function requires the F-box domain of FBXO16, through which FBXO16 assembles a canonical SCF ubiquitin ligase complex that constitutively targets hnRNPL for degradation. Depletion of hnRNPL is sufficient to inactive multiple oncogenic signaling regulated by FBXO16 and prevent the malignant behavior of ovarian cancer cells caused by FBXO16 deficiency. FBXO16 interacted with the RRM3 domain of hnRNPL via its C-terminal region to trigger the proteasomal degradation of hnRNPL. Failure to degrade hnRNPL promoted ovarian cancer cell proliferation in vitro and tumor growth vivo, phenocopying the deficiency of FBXO16 in ovarian cancer.Subject terms: Ovarian cancer, Oncogenes     

E-cadherin's dark side: Possible role in tumor progression

In the context of cancer, E-cadherin has traditionally been categorized as a tumor suppressor, given its essential role in the formation of proper intercellular junctions, and its downregulation in the process of epithelial–mesenchymal transition (EMT) in epithelial tumor progression. Germline or somatic mutations in the E-cadherin gene (CDH1) or downregulation by epigenetic mechanisms have been described in a small subset of epithelial cancers. However, recent evidence also points toward a promoting role of E-cadherin in several aspects of tumor progression. This includes preserved (or increased) E-cadherin expression in microemboli of inflammatory breast carcinoma, a possible “mesenchymal to epithelial transition” (MET) in ovarian carcinoma, collective cell invasion in some epithelial cancers, a recent association of E-cadherin expression with a more aggressive brain tumor subset, as well as the intriguing possibility of E-cadherin involvement in specific signaling networks in the cytoplasm and/or nucleus. In this review we address a lesser-known, positive role for E-cadherin in cancer.     

Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized β-transducin repeat-containing protein 1 (βTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.     

Distinct Patterns of Stromal and Tumor Expression of ROR1 and ROR2 in Histological Subtypes of Epithelial Ovarian Cancer

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n = 178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.