Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies.

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels

Intrinsic differences between authentic and cryptic 5 ' splice sites

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5' splice sites (5'ss), we systematically analyzed a set of 76 cryptic 5'ss derived from 46 human genes. These cryptic 5'ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5'ss. Statistical analysis of the strengths of the 5'ss using the Shapiro and Senapathy matrix revealed that authentic 5'ss have significantly higher score values than cryptic 5'ss, which in turn have higher values than the mutant ones. beta-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the beta-globin authentic and cryptic 5'ss, but not their surrounding context, determine the correct 5'ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5'ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5'ss-disrupting mutations at the mRNA level

UGT genomic diversity : beyond gene duplication

The human uridine diphospho (UDP)-glucuronosyltransferase (UGT) superfamily comprises enzymes responsible for a major biotransformation phase II pathway: the glucuronidation process. The UGT enzymes are located in the endoplasmic reticulum of almost all tissues, where they catalyze the inactivation of several endogenous and exogenous molecules, including bilirubin, sex steroids, numerous prescribed drugs, and environmental toxins. This metabolic pathway is particularly variable. The influence of inheritable polymorphisms in human UGT-encoding genes has been extensively documented and was shown to be responsible for a fraction of the observed phenotypic variability. Other key genomic processes are likely underlying this diversity; these include copy-number variations, epigenetic factors, and newly discovered splicing mechanisms. This review will discuss novel molecular aspects that may be determinant to UGT phenotypes

Comprehensive assessment of cancer missense mutation clustering in protein structures

Large-scale tumor sequencing projects enabled the identification of many new cancer gene candidates through computational approaches. Here, we describe a general method to detect cancer genes based on significant 3D clustering of mutations relative to the structure of the encoded protein products. The approach can also be used to search for proteins with an enrichment of mutations at binding interfaces with a protein, nucleic acid, or small molecule partner. We applied this approach to systematically analyze the PanCancer compendium of somatic mutations from 4,742 tumors relative to all known 3D structures of human proteins in the Protein Data Bank. We detected significant 3D clustering of missense mutations in several previously known oncoproteins including HRAS, EGFR, and PIK3CA. Although clustering of missense mutations is often regarded as a hallmark of oncoproteins, we observed that a number of tumor suppressors, including FBXW7, VHL, and STK11, also showed such clustering. Beside these known cases, we also identified significant 3D clustering of missense mutations in NUF2, which encodes a component of the kinetochore, that could affect chromosome segregation and lead to aneuploidy. Analysis of interaction interfaces revealed enrichment of mutations in the interfaces between FBXW7-CCNE1, HRAS-RASA1, CUL4B-CAND1, OGT-HCFC1, PPP2R1A-PPP2R5C/PPP2R2A, DICER1-Mg 2+ , MAX-DNA, SRSF2-RNA, and others. Together, our results indicate that systematic consideration of 3D structure can assist in the identification of cancer genes and in the understanding of the functional role of their mutations. Keywords: cancer; cancer genetics; mutation clustering; protein structures; interaction interfacesNational Institutes of Health (U.S.) (Grant U24 CA143845

Alternative Splicing and Tumor Progression

Alternative splicing is a key molecular mechanism for increasing the functional diversity of the eukaryotic proteomes. A large body of experimental data implicates aberrant splicing in various human diseases, including cancer. Both mutations in cis-acting splicing elements and alterations in the expression and/or activity of splicing regulatory factors drastically affect the splicing profile of many cancer-associated genes. In addition, the splicing profile of several cancer-associated genes is altered in particular types of cancer arguing for a direct role of specific splicing isoforms in tumor progression. Deciphering the mechanisms underlying aberrant splicing in cancer may prove crucial to understand how splicing machinery is controlled and integrated with other cellular processes, in particular transcription and signaling pathways. Moreover, the characterization of splicing deregulation in cancer will lead to a better comprehension of malignant transformation. Cancer-associated alternative splicing variants may be new tools for the diagnosis and classification of cancers and could be the targets for innovative therapeutical interventions based on highly selective splicing correction approaches

The effect of hypoxia on alternative splicing in prostate cancer cell lines

Hypoxia is defined as the state in which the availability or delivery of oxygen is insufficient to meet tissue demand. It occurs particularly in aggressive, fast-growing tumours in which the rate of new blood vessel formation (angiogenesis) cannot match the growth rate of tumour cells. Cellular stresses such as hypoxia can cause cells to undergo apoptosis; however some tumour cells adapt to hypoxic conditions and evade apoptosis. Tumour hypoxia has been linked to poor prognosis and to greater resistance to existing cancer therapies. This thesis provides evidence that alterations in alternative splicing patterns of key genes is one method tumour cells adapt to hypoxia.This study confirms a hypoxic-induced change in the alternative splicing of carbonic anhydrase IX (CA IX) following 1% oxygen treatment. CA IX is one of the best studied hypoxia markers, involved in maintaining an intracellular pH that favours tumour cell growth. Furthermore, evidence is provided here that in PC3 cells the regulation of CA IX splicing involves the SAFB1 and PRPF8 splice factors. Additionally, SAFB1 expression is shown to decrease in hypoxia. This study further demonstrates that alternative splicing patterns of previously documented cancer-associated genes are altered in hypoxia. PCR analysis showed that hypoxia significantly altered the alternative splicing of apoptotic-associated genes: caspase-9; Mcl-1; Bcl-x; survivin. The expression of the pro-apoptotic isoforms of the first two genes, and the anti-apoptotic isoforms of the latter two genes were favoured by hypoxia. Furthermore, high-throughput PCR analysis provided evidence of significant changes in the alternative splicing of several other cancer-associated genes in hypoxia: APAF1; BTN2A2; CDC42BPA; FGFR1OP; MBP; PTPN13; PUF60; RAP1GDS1; TTC23; UTRN. Most notably, the pro-oncogenic isoforms of APAF1, BTN2A2 and RAP1GDS1 were favoured in hypoxia. The majority of alternative splicing changes were found in the PC3 cell line. However changes in alternative splicing patterns that mirrored those in the PC3 cell line were also found in the VCaP (CDC42BPA, RAP1GDS1 and UTRN) and PNT2 (BTN2A2, CDC42BPA, FGFR1OP and TTC23) cell lines. The mRNA expression of splice factors (SRSF1, SRSF2, SRSF3, SAM68, HuR and hnRNP A1) and splice factor kinases (CLK1 and SRPK1) were shown to significantly increase in hypoxia. Subsequent experiments provided evidence that CLK1 and SRSF1 protein expression also increased in hypoxia. The phosphorylation of SRSF4 and SRSF5 were demonstrated to increase in hypoxia. However, the phosphorylation of SRSF6 was not. In addition, siRNAs and chemical inhibitors of CLK1 (TG003) and SRPK1 (SPHINX) were used to assess the effect of these splice factor kinases on the subsequent splicing of cancer-associated genes. There were no significant changes to splicing found with SRPK1 siRNA knockdown or SPHINX treatment. However CLK1 siRNA knockdown and TG003 treatment demonstrated a shift in FGFR1OP splicing that mirrored the effect of hypoxia on FGFR1OP splicing. This suggests that CLK1 activity is inhibited in hypoxia. Furthermore, in contrast to previous research CLK1 was found to be localised to the cytoplasm in both normoxia and hypoxia in the PC3 cell line. This work has uncovered factors and provided an insight into mechanisms that are involved in alternative splicing changes in hypoxia in mammalian cell lines. It is hoped that these novel research findings will aid in the understanding of how cells adapt to hypoxia especially in regards to alternative splicing, and may offer future therapeutic targets in hypoxic tumours