Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3'UTRs, including those involved in the transforming growth factor-ß signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.

3' UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk.

Widespread mRNA 3' UTR shortening through alternative polyadenylation 1 promotes tumor growth in vivo 2 . A prevailing hypothesis is that it induces proto-oncogene expression in cis through escaping microRNA-mediated repression. Here we report a surprising enrichment of 3'UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor-suppressor genes. Our model-based analysis of the trans effect of 3' UTR shortening (MAT3UTR) reveals a significant role in altering ceRNA expression. MAT3UTR predicts many trans-targets of 3' UTR shortening, including PTEN, a crucial tumor-suppressor gene 3 involved in ceRNA crosstalk 4 with nine 3'UTR-shortening genes, including EPS15 and NFIA. Knockdown of NUDT21, a master 3' UTR-shortening regulator 2 , represses tumor-suppressor genes such as PHF6 and LARP1 in trans in a miRNA-dependent manner. Together, the results of our analysis suggest a major role of 3' UTR shortening in repressing tumor-suppressor genes in trans by disrupting ceRNA crosstalk, rather than inducing proto-oncogenes in cis.

Molecular targeting of glutaminase sensitizes ovarian cancer cells to chemotherapy.

Altered metabolism is a reemerging hallmark of tumorigenesis. Increased cell proliferation results in metabolic reprogramming to facilitate the needs of the rapidly dividing tumor cells. In addition to increased glucose uptake, tumors also take up increased levels of glutamine. Some cancers develop a reliance on glutamine, and are referred to as "glutamine addicted." These tumors over express the enzyme glutaminase which is involved in the first step of glutaminolysis. The goal of this study was to determine the effects of combined treatment of the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) with chemotherapy on drug resistant ovarian cancer cells. We found that ovarian cancer cells show different dependencies on exogenous glutamine. However, regardless of glutamine dependence status, treatment with BPTES sensitized both paclitaxel, and cisplatin resistant cancer cell lines to chemotherapy by inhibiting cell proliferation. Monotherapy with BPTES alone resulted in a significant reduction in the ability of glutamine dependent cancer cells to form colonies in a clonogenic assay. In addition, glutamine dependent, metastatic cancer cells expressed higher levels of glutaminase 1 (GLS1) isoforms, KGA and GAC, than untransformed cells. Moreover, dual targeting of both isoforms using siRNA was more effective at sensitizing the cancer cells to cisplatin than targeting either GAC or KGA alone. Our results suggest that both GLS1 isoforms are important for glutamine dependent ovarian cancer survival, hence, both GLS1 isoforms should be targeted for therapy in metastatic ovarian cancer therapy.

The contribution of alternative polyadenylation to the cancer phenotype.

Eukaryotic 3'-end formation is a critical step for mRNA maturation and involves a requisite cleavage and polyadenylation event downstream of a polyadenylation signal. Recent discoveries suggest that in nearly 70% of human genes, cleavage and polyadenylation can occur at multiple locations through a process known as alternative polyadenylation (APA). Therefore, APA is a form of co-transcriptional gene regulation that has the potential to greatly expand mRNA transcript diversity. There are two general types of APA. The first includes alternative splicing events (splicing-APA) that result in changes to the coding sequence, and the second does not involve alternative splicing (tandem untranslated region-APA). The latter form of APA occurs within the same terminal exon, thereby only changing the 3'-untranslated region length and content. The role of APA in transformation and cancer is still being deciphered and has been the subject of intensive investigation by multiple groups. Here, we provide a general summary of APA and how it can differentially impact mRNA stability, translation and localization in cis. In addition, we discuss more indirect implications of APA on mRNA transcripts. The latter is based upon the concept that APA can induce the reorganization of both microRNA-mRNA and RNA-binding protein-mRNA interactions. We use these general models as a platform to describe what is known about how tumors manipulate the APA of oncogenes to further drive tumorigenesis. Finally, we briefly discuss the role that next-generation sequencing has played in identifying APA regulators and key transcripts that alter 3'-untranslated region length in cancer.

Development of a dietary formulation of the SHetA2 chemoprevention drug for mice.

Development of cancer chemoprevention compounds requires enhanced consideration for toxicity and route of administration because the target population is healthy. The small molecule drug, SHetA2 (NSC 726189), exhibited in vivo chemoprevention activity and lack of toxicity when administered by oral gavage. Our objective was to determine if a dietary formulation of SHetA2 could achieve effective tissue drug levels without toxicity. C57bl/6 J mice were monitored on modified American Institute of Nutrition (AIN)76A diet mixed with SHetA2 in a 3:1 ratio with Kolliphor HS15, a self-emulsifying drug delivery system (SEDDS) to deliver 37.5, 62.5, 125, 187 or 250 mg SHetA2/kg/day. Blood and tissues were evaluated after 1, 3 and 6 weeks. The 187 mg/kg/day dose was identified as optimal based on achievement of maximum blood and tissue drug levels in the effective micromolar range without evidence of toxicity. The 250 mg/kg/day group exhibited lower drug levels and the highest intestinal drug content suggesting that an upper limit of intestinal absorption had been surpassed. Only this highest dose resulted in liver and kidney function tests that were outside of the normal range, and significant reduction of cyclin D1 protein in normal cervical tissue. SHetA2 reduced cyclin D1 to greater extents in cancer compared to non-cancer cell cultures. Given this differential effect, optimal chemoprevention without toxicity would be expected to occur at doses that reduced cyclin D1 in neoplastic, but not in normal tissues. These findings support further development of SHetA2 as a chemoprevention agent and potential food additive.

CFIm25 regulates glutaminase alternative terminal exon definition to modulate miR-23 function.

Alternative polyadenylation (APA) and alternative splicing (AS) provide mRNAs with the means to avoid microRNA repression through selective shortening or differential usage of 3'UTRs. The two glutaminase (GLS) mRNA isoforms, termed KGA and GAC, contain distinct 3'UTRs with the KGA isoform subject to repression by miR-23. We show that depletion of the APA regulator CFIm25 causes a strong shift to the usage of a proximal poly(A) site within the KGA 3'UTR and also alters splicing to favor exclusion of the GAC 3'UTR. Surprisingly, we observe that while miR-23 is capable of down-regulating the shortened KGA 3'UTR, it has only minor impact on the full-length KGA 3'UTR, demonstrating that additional potent negative regulation of GLS expression exists beyond this single microRNA targeting site. Finally, we show that the apoptosis induced upon down-regulation of the GAC isoform can be alleviated through concurrent reduction in CFIm25 expression, revealing the sensitivity of glutaminase expression to the levels of RNA processing factors. These results exemplify the complex interplay between RNA processing and microRNA repression in controlling glutamine metabolism in cancer cells.

CFIm25 links alternative polyadenylation to glioblastoma tumour suppression.

The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3' untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3'-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3' UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3' UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.