S. Duygu Selcuklu

miR-21 as a key regulator of oncogenic processes

Small non-coding miRNAs (microRNAs) are emerging as key factors involved in cancer at all stages ranging from initiation to metastasis. MIRN21 is an miRNA gene that codes for the miR-21 miRNA which has been found to be overexpressed in many tumour samples where it has been analysed. Whereas consistent overexpression of miR-21 in tumours could be suggestive of functional effects of miR-21 in cancer, more in-depth functional studies with miR-21 are demonstrating that mir-21 displays oncogenic activity and can be classed as an oncomir. Extensive efforts are underway to identify the downstream genes and gene networks regulated by miR-21 and to identify the upstream factors that are regulating expression of miR-21. Even though miR-21 is one of the most intensively studied miRNAs, for all miRNAs, our understanding of miRNA signalling pathways is currently in its early stages. The unravelling of such RNA signalling pathways and networks will be key to understanding the role that dysregulated miRNA functioning can play in oncogenic processes.

MicroRNA-9 Inhibition of Cell Proliferation and Identification of Novel miR-9 Targets by Transcriptome Profiling in Breast Cancer Cells

Background: Dysregulation of miRNAs is associated with breast cancer. Results: MiR-9 overexpression and transcriptome analysis reveals novel miR-9 targets, including MTHFD2, which can recapitulate anti-proliferative effects of miR-9 overexpression. Conclusion: MiR-9 displays tumor suppressor-like activity in breast cancer cells; MTHFD2 contributes to this activity. Significance: Understanding miR-9-directed regulation of the breast cancer transcriptome is important for diagnosis and therapeutics. Although underexpression of miR-9 in cancer cells is reported in many cancer types, it is currently difficult to classify miR-9 as a tumor suppressor or an oncomir. We demonstrate that miR-9 expression is down-regulated in MCF-7 and MDA-MB-231 breast cancer cells compared with MCF-10-2A normal breast cell line. Increasing miR-9 expression levels in breast cancer cells induced anti-proliferative, anti-invasive, and pro-apoptotic activity. In addition, microarray profiling of the transcriptome of MCF-7 cells overexpressing miR-9 identified six novel direct miR-9 targets (AP3B1, CCNG1, LARP1, MTHFD1L, MTHFD2, and SRPK1). Among these, MTHFD2 was identified as a miR-9 target gene that affects cell proliferation. Knockdown of MTHFD2 mimicked the effect observed when miR-9 was overexpressed by decreasing cell viability and increasing apoptotic activity. Despite variable effects on different cell lines, proliferative and anti-apoptotic activity of MTHFD2 was demonstrated whereby it could escape from miR-9-directed suppression (by overexpression of MTHFD2 with mutated miR-9 binding sites). Furthermore, endogenous expression levels of miR-9 and MTHFD2 displayed inverse expression profiles in primary breast tumor samples compared with normal breast samples; miR-9 was down-regulated, and MTHFD2 was up-regulated. These results indicate anti-proliferative and pro-apoptotic activity of miR-9 and that direct targeting of MTHFD2 can contribute to tumor suppressor-like activity of miR-9 in breast cancer cells.

Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid

Purpose Cancer spread to the central nervous system (CNS) often is diagnosed late and is unresponsive to therapy. Mechanisms of tumor dissemination and evolution within the CNS are largely unknown because of limited access to tumor tissue. Materials and Methods We sequenced 341 cancer-associated genes in cell-free DNA from cerebrospinal fluid (CSF) obtained through routine lumbar puncture in 53 patients with suspected or known CNS involvement by cancer. Results We detected high-confidence somatic alterations in 63% (20 of 32) of patients with CNS metastases of solid tumors, 50% (six of 12) of patients with primary brain tumors, and 0% (zero of nine) of patients without CNS involvement by cancer. Several patients with tumor progression in the CNS during therapy with inhibitors of oncogenic kinases harbored mutations in the kinase target or kinase bypass pathways. In patients with glioma, the most common malignant primary brain tumor in adults, examination of cell-free DNA uncovered patterns of tumor evolution, including temozolomide-associated mutations. Conclusion The study shows that CSF harbors clinically relevant genomic alterations in patients with CNS cancers and should be considered for liquid biopsies to monitor tumor evolution in the CNS.

Genomic Biomarkers of a Randomized Trial Comparing First-line Everolimus and Sunitinib in Patients with Metastatic Renal Cell Carcinoma

BACKGROUND Metastatic renal cell carcinoma (RCC) patients are commonly treated with vascular endothelial growth factor (VEGF) inhibitors or mammalian target of rapamycin inhibitors. Correlations between somatic mutations and first-line targeted therapy outcomes have not been reported on a randomized trial. OBJECTIVE To evaluate the relationship between tumor mutations and treatment outcomes in RECORD-3, a randomized trial comparing first-line everolimus (mTOR inhibitor) followed by sunitinib (VEGF inhibitor) at progression with the opposite sequence in 471 metastatic RCC patients. DESIGN, SETTING, AND PARTICIPANTS Targeted sequencing of 341 cancer genes at ∼540× coverage was performed on available tumor samples from 258 patients; 220 with clear cell histology (ccRCC). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Associations between somatic mutations and median first-line progression free survival (PFS1L) and overall survival were determined in metastatic ccRCC using Cox proportional hazards models and log-rank tests. RESULTS AND LIMITATIONS Prevalent mutations (≥ 10%) were VHL (75%), PBRM1 (46%), SETD2 (30%), BAP1 (19%), KDM5C (15%), and PTEN (12%). With first-line everolimus, PBRM1 and BAP1 mutations were associated with longer (median [95% confidence interval {CI}] 12.8 [8.1, 18.4] vs 5.5 [3.1, 8.4] mo) and shorter (median [95% CI] 4.9 [2.9, 8.1] vs 10.5 [7.3, 12.9] mo) PFS1L, respectively. With first-line sunitinib, KDM5C mutations were associated with longer PFS1L (median [95% CI] of 20.6 [12.4, 27.3] vs 8.3 [7.8, 11.0] mo). Molecular subgroups of metastatic ccRCC based on PBRM1, BAP1, and KDM5C mutations could have predictive values for patients treated with VEGF or mTOR inhibitors. Most tumor DNA was obtained from primary nephrectomy samples (94%), which could impact correlation statistics. CONCLUSIONS PBRM1, BAP1, and KDM5C mutations impact outcomes of targeted therapies in metastatic ccRCC patients. PATIENT SUMMARY Large-scale genomic kidney cancer studies reported novel mutations and heterogeneous features among individual tumors, which could contribute to varied clinical outcomes. We demonstrated correlations between somatic mutations and treatment outcomes in clear cell renal cell carcinoma, supporting the value of genomic classification in prospective studies.

An NTD‐Associated Polymorphism in the 3′ UTR of MTHFD1L can Affect Disease Risk by Altering miRNA Binding

Maternal folate levels and polymorphisms in folate‐related genes are known risk factors for neural tube defects (NTDs). SNPs in the mitochondrial folate gene MTHFD1L are associated with the risk of NTDs. We investigated whether different alleles of SNP rs7646 in the 3′ UTR of MTHFD1L can be differentially regulated by microRNAs affecting MTHFD1L expression. We previously reported that miR‐9 targets MTHFD1L and now we identify miR‐197 as an additional miRNA regulator. Both of these miRNAs have predicted binding sites in the MTHFD1L 3′ UTR in the region containing SNP rs7646. We have determined whether the alleles of SNP rs7646 (A/G) and miRNA expression levels affect miRNA binding preferences for the MTHFD1L 3′ UTR and consequently MTHFD1L expression. Our results indicate that miR‐9 and miR‐197 specifically downregulate MTHFD1L levels in HEK293 and MCF‐7 cells and that SNPrs7646 significantly affects miR‐197 binding affinity to the MTHFD1L 3′ UTR, causing more efficient posttranscriptional gene repression in the presence of the allele that is associated with increased risk of NTDs. These results reveal that the association of SNP rs7646 and NTD risk involves differences in microRNA regulation and, highlights the importance of genotype‐dependent differential microRNA regulation in relation to human disease risk.

Phase II Trial and Correlative Genomic Analysis of Everolimus Plus Bevacizumab in Advanced Non–Clear Cell Renal Cell Carcinoma

Purpose The decreased effectiveness of single-agent targeted therapies in advanced non-clear cell renal cell carcinoma (ncRCC) compared with clear cell renal cell carcinoma (RCC) supports the study of combination regimens. We evaluated the efficacy of everolimus plus bevacizumab in patients with metastatic ncRCC. Patients and Methods In this single-center phase II trial, treatment-naive patients received everolimus 10 mg oral once per day plus bevacizumab 10 mg/kg intravenously every 2 weeks. The primary end point was progression-free survival (PFS) at 6 months. Correlative analyses explored candidate tissue biomarkers through next-generation sequencing. Results Thirty-five patients were enrolled with the following histologic subtypes: chromophobe (n = 5), papillary (n = 5), and medullary (n = 2) RCC and unclassified RCC (uRCC, n = 23). The majority of patients had papillary growth as a major component (n = 14). For 34 evaluable patients, median PFS, overall survival, and objective response rate (ORR) were 11.0 months, 18.5 months, and 29%, respectively. PFS varied by histology ( P < .001), and ORR was higher in patients with significant papillary (seven of 18) or chromophobe (two of five) elements than for others (one of 11). Presence of papillary features were associated with benefit, including uRCC, where it correlated with ORR (43% v 11%), median PFS (12.9 v 1.9 months), and overall survival (28.2 v 9.3 months; P < .001). Several genetic alterations seemed to segregate by histology. In particular, somatic mutations in ARID1A were seen in five of 14 patients with papillary features but not in other RCC variants. All five patients achieved treatment benefit. Conclusion The study suggests efficacy for this combination in patients with ncRCC characterized by papillary features. Distinct mutational profiles among ncRCCs vary according to specific histology.

Translational Epigenetics: Clinical Approaches to Epigenome Therapeutics for Cancer

Cancer epigenetics research is now entering an exciting phase of translational epigenetics whereby novel epigenome therapeutics is being developed for application in clinical settings. Epigenetics refers to all heritable and potentially reversible changes in gene or genome functioning that occurs without altering the nucleotide sequence of the DNA. A range of different epigenetic “marks” can activate or repress gene expression. While epigenetic alterations are associated with most cancers, epigenetic dysregulation can also have a causal role in cancer etiology. Epigenetically disrupted stem or progenitor cells could have an early role in neoplastic transformations, while perturbance of epigenetic regulatory mechanisms controlling gene expression in cancer-relevant pathways will also be a contribution factor. The reversibility of epigenetic marks provides the possibility that the activity of key cancer genes and pathways can be regulated as a therapeutic approach. The growing availability of a range of chemical agents which can affect epigenome functioning has led to a range of epigenetic-therapeutic approaches for cancer and intense interest in the development of second-generation epigenetic drugs (epi-drugs) which would have greater specificity and efficacy in clinical settings. The latest developments in this exciting arena of translational cancer epigenetics were presented at a recent conference on “Epigenetics and New Therapies in Cancer” at the Spanish National Cancer Research Center (CNIO), Spain.

ALCAM is indirectly modulated by miR-125b in MCF7 cells

MicroRNA (miRNA) deregulation is associated with various cancers. Among an expanding list of cancer-related miRNAs, deregulation of miR-125b has been well documented in many cancers including breast. Based on current knowledge, miR-125b is considered to be a tumor suppressor in breast cancers. While important messenger RNA (mRNA) targets have been defined for miR-125b, here, we aimed to further investigate direct/indirect consequences of miR-125b expression in breast cancer cells by using a transcriptome approach. Upon miR-125b expression, a total of 138 cancer-related genes were found to be differentially expressed in breast cancer cells. While only a few of these were predicted to be direct mRNA targets, majority of the gene expression changes were potentially downstream and indirect effects of miR-125b expression. Among these, activated leukocyte antigen molecule (ALCAM) mRNA and protein levels were found to be highly significantly increased upon miR-125b expression. Given the tumor suppressor role of miR-125b in our model system, upon silencing of ALCAM expression, cell proliferation rate re-increased in miR-125b-expressing cells. While ALCAM’s possible context-dependent roles are not clear in breast cancer, a diverse expression pattern of ALCAM mRNA was detected in a panel of breast cancer patient samples. Differentially expressed/regulated cancer-related genes upon miR-125b expression along with the significant increase of ALCAM are of future interest to understand how deregulated expression of miR-125b may have a tumor suppressor role in breast and other cancers.

Allele-specific splicing effects on DKKL1 and ZNF419 transcripts in HeLa cells.

Allele-specific splicing is the production of different RNA isoforms from different alleles of a gene. Altered splicing patterns such as exon skipping can have a dramatic effect on the final protein product yet have traditionally proven difficult to predict. We investigated the splicing effects of a set of nine single nucleotide polymorphisms (SNPs) which are predicted to have a direct impact on mRNA splicing, each in a different gene. Predictions were based on SNP location relative to splice junctions and intronic/exonic splicing elements, combined with an analysis of splice isoform expression data from public sources. Of the nine genes tested, six SNPs led to direct impacts on mRNA splicing as determined by the splicing reporter minigene assay and RT-PCR in human HeLa cells, of which four were allele-specific effects. These included previously unreported alternative splicing patterns in the genes ZNF419 and DKKL1. Notably, the SNP in ZNF419, a transcription factor, leads to the deletion of a DNA-binding domain from the protein and is associated with an expression QTL, while the SNP in DKKL1 leads to shortened transcripts predicted to produce a truncated protein. We conclude that the impact of SNP mutations on mRNA splicing, and its biological relevance, can be predicted by integrating SNP position with available data on relative isoform abundance in human cell lines.