Pre-mRNA processing factor 3 enhances the progression of keratinocyte-derived cutaneous squamous cell carcinoma by regulating the JAK2/STAT3 pathway.

The precise role of pre-mRNA processing factors (PRPs) in human tumorigenesis has not been yet explored. The object of the present study was to explore the effects of PRP3 in a common metastatic skin cancer, keratinocyte-derived cutaneous squamous cell carcinoma (cSCCs). RT-qPCR and western blotting were conducted to measure the expression levels of PRP3 in various cSCC cell lines and cSCC tissues. A benign epidermal keratinocyte cell line was transfected with a eukaryotic expression plasmid to overexpress PRP3. In addition, the endogenous expression level of PRP3 in cSCC cells was silenced using a short hairpin RNA method, and the role of PRP3 on cell proliferation and migration was examined by Cell Counting Kit-8, colony formation, wound healing assay and Transwell assays following knockdown in cSCC cells, and overexpression in keratinovcyte cells. Elevated levels of PRP3 mRNA and protein were noted in cSCC cell lines or cSCC tissues compared with actinic keratosis (AK) or benign epidermal keratinocyte cell line, respectively. Upregulation of PRP3 expression was found to be associated with poor clinical outcomes in patients with cSCCs. The upregulation of PRP3 promoted cell viability, metastasis and the activity of the JAK2/STAT3 pathway in epidermal keratinocyte cells. Interestingly, loss of PRP3 had no obvious impact on cell viability and migration in benign epidermal keratinocyte cells. Functionally, the inhibition of the JAK2/STAT3 pathway reversed the increased cell viability and migration of cSCC cells induced by PRP3. Taken together, the present observations indicated that PRP3 served as a tumor active factor in cSCCs by targeting the JAK2/STAT3 pathway. Moreover, it is implied that impeding the PRP3 activity may selectively constrain cancer cell growth and migration with limited effect on normal skin cells.

Suppression of PRPF4 regulates pluripotency, proliferation, and differentiation in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are characterized by their self-renewal and pluripotency and are capable of differentiating into all three germ layers. For this reason, mESCs are considered a very important model for stem cell research and clinical applications in regenerative medicine. The pre-mRNA processing factor 4 (PRPF4) gene is known to have a major effect on pre-mRNA splicing and is also known to affect tissue differentiation during development. In this study, we investigated the effects of PRPF4 knockdown on mESCs. First, we allowed mESCs to differentiate naturally and observed a significant decrease in PRPF4 expression during the differentiation process. We then artificially induced the knockdown of PRPF4 in mESCs and observed the changes in the phenotype. When PRPF4 was knocked down, various genes involved in mESC pluripotency showed significantly decreased expression. In addition, mESC proliferation increased abnormally, accompanied by a significant increase in mESC colony size. The formation of mESC embryoid bodies and teratomas was delayed following PRPF4 knockdown. Based on these results, the reduced expression of PRPF4 affects mESC phenotypes and is a key factor in mESC. SIGNIFICANCE OF THE STUDY: Our results indicate that PRPF4 affects the properties of mESCs. Suppression of PRPF4 resulted in a decrease in pluripotency of mESC and promoted proliferation. In addition, suppression of PRPF4 also resulted in decreased apoptosis. Moreover, the inhibition of PRPF4 reduced the ability to differentiate and formation of teratoma in mESC. Our results demonstrated that PRPF4 is a key factor of controlling mESC abilities.

Estrogen receptor alpha (ESR1)-signaling regulates the expression of the taxane-response biomarker PRP4K.

The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), is an essential kinase that is a component of the U5 snRNP and functions in spliceosome assembly. We demonstrated that PRP4K is a novel biological marker for taxane response in ovarian cancer patients and reduced levels of PRP4K correlate with intrinsic and acquired taxane resistance in both breast and ovarian cancer. Breast cancer treatments are chosen based on hormone and growth factor receptor status, with HER2 (ERBB2) positive breast cancer patients receiving anti-HER2 agents and taxanes and estrogen receptor alpha (ESR1) positive (ER+) breast cancer patients receiving anti-estrogen therapies such as tamoxifen. Here we demonstrate that PRP4K is expressed in the normal mammary duct epithelial cells of the mouse, and that estrogen induces PRP4K gene and protein expression in ER+ human MCF7 breast cancer cells. Estrogen acts through ESR1 to regulate PRP4K expression, as over-expression of ESR1 in the ER-negative MDA-MB-231 breast cancer cell line increased the expression of this kinase, and knock-down of ESR1 in ER+ T47D breast cancer cells reduced PRP4K levels. Furthermore, treatment with 4-hydroxytamoxifen (4-OHT) resulted in a dose-dependent decrease in PRP4K protein expression in MCF7 cells. Consistent with our previous studies identifying PRP4K as a taxane-response biomarker, reduced PRP4K expression in 4-OHT-treated cells correlated with reduced sensitivity to paclitaxel. Thus, PRP4K is novel estrogen regulated kinase, and its levels can be reduced by 4-OHT in ER+ breast cancer cells altering their response to taxanes.

Curcumin induces radiosensitivity of in vitro and in vivo cancer models by modulating pre-mRNA processing factor 4 (Prp4).

Radiation therapy plays a central role in adjuvant strategies for the treatment of both pre- and post-operative human cancers. However, radiation therapy has low efficacy against cancer cells displaying radio-resistant phenotypes. Ionizing radiation has been shown to enhance ROS generation, which mediates apoptotic cell death. Further, concomitant use of sensitizers with radiation improves the efficiency of radiotherapy against a variety of human cancers. Here, the radio-sensitizing effect of curcumin (a derivative of turmeric) was investigated against growth of HCT-15 cells and tumor induction in C57BL/6J mice. Ionizing radiation induced apoptosis through ROS generation and down-regulation of Prp4K, which was further potentiated by curcumin treatment. Flow cytometry revealed a dose-dependent response for radiation-induced cell death, which was remarkably reversed by transfection of cells with Prp4K clone. Over-expression of Prp4K resulted in a significant decrease in ROS production possibly through activation of an anti-oxidant enzyme system. To elucidate an integrated mechanism, Prp4K knockdown by siRNA ultimately restored radiation-induced ROS generation. Furthermore, B16F10 xenografts in C57BL/6J mice were established in order to investigate the radio-sensitizing effect of curcumin in vivo. Curcumin significantly enhanced the efficacy of radiation therapy and reduced tumor growth as compared to control or radiation alone. Collectively, these results suggest a novel mechanism for curcumin-mediated radio-sensitization of cancer based on ROS generation and down-regulation of Prp4K.

Evaluation of cancer dependence and druggability of PRP4 kinase using cellular, biochemical, and structural approaches.

PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.

The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly.

Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev binds to a cis-acting Rev-responsive element (RRE) located within the env region of HIV-1. It has previously been shown that a 17-amino-acid peptide, corresponding to the basic domain of Rev, specifically inhibited in vitro the splicing of mRNAs containing the RRE. In this reaction, the peptide acts after an ATP-dependent step in the spliceosome assembly resulting in an accumulation of a 45-50S splicing-deficient complex. Characterization of this complex revealed that the basic domain of Rev does not interfere with U1 small nuclear ribonucleoprotein binding but blocks the entry of U4, U5, and U6 small nuclear RNAs into the spliceosome. Binding of U2 small nuclear ribonucleoprotein was partially inhibited. The critical nature of the oligomeric structure of RRE has been investigated both in vitro and in vivo. Reporter genes that contained one, three, or six repeated-monomer high-affinity Rev binding sites (IIB) within an intron yielded a correlation among the oligomeric state of bound Rev; inhibition of splicing; ability to block the assembly of U4, U5, and U6 small nuclear RNAs in the spliceosome in vitro; and level of Rev response in vivo.