Elucidating the mechanisms involved in sensitizing radioresistant tumors to ionizing radiation (IR) treatments while minimizing injury to surrounding normal tissue is an important clinical goal. Due ...to their sequence-derived specificity and properties as gene regulators in IR-affected pathways, microRNAs (miRNAs) could serve as adjuvant therapeutic agents that alter cellular sensitivity to radiation treatment. To identify radiosensitizing miRNAs, we initially utilized the Caenorhabditis elegans vulval cell model, an in vivo system developed to study IR-dependent radiosensitivity as a measure of clonogenic cell death. We tested several candidate miRNA-deletion mutants post γ-irradiation and identified cel-mir-237 as a miRNA which when deleted caused animals to be more resistant to IR, whereas cel-mir-237 overexpressing strains were IR sensitive. In addition, wild-type animals downregulated cel-mir-237 levels post IR in a time-dependent manner. We identified jun-1 (JUN transcription factor homolog) as a novel target of cel-mir-237. Specifically, jun-1 transcript levels increased in wild-type animals post γ-irradiation, and loss of cel-mir-237 also resulted in higher jun-1 expression. As expected, loss of jun-1 resulted in IR sensitivity, similar to the phenotype of cel-mir-237 overexpressors. As miR-237 is the homolog of human miR-125, we validated our findings in MCF-7 and MDA-MB-231 breast cancer cell lines, which harbor lower hsa-miR-125b levels than normal human mammary epithelial cells (HMECs). Forced expression of hsa-miR-125b in these cells resulted in radiosensitivity, as seen by reduced clonogenic survival, enhanced apoptotic activity and enhanced senescence post IR. Finally, re-expression of c-JUN in MDA-MB-231 cells promoted radioresistance and abrogated miR-125-mediated radiosensitization. Our findings suggest that overexpression of cel-mir-237 and its homolog, hsa-miR-125b, functions as sensitizers to γ-irradiation in both a nematode in vivo model and breast cancer cells, and could potentially be utilized as an adjuvant therapeutic to enhance radiation sensitivity.
MicroRNAs (miRNAs) are important regulators of cell fate determination and homeostasis. Expression of these small RNA genes is tightly regulated during development and in normal tissues, but they are ...often misregulated in cancer. MiRNA expression is also affected by DNA damaging agents, such as radiation. In particular, mammalian miR-34 is upregulated by p53 in response to radiation, but little is known about the role of this miRNA in vivo. Here we show that Caenorhabditis elegans with loss-of-function mutations in the mir-34 gene have an abnormal cellular survival response to radiation; these animals are highly radiosensitive in the soma and radioresistant in the germline. These findings show a role for mir-34 in both apoptotic and non-apoptotic cell death in vivo, much like that of cep-1, the C. elegans p53 homolog. These results have been additionally validated in vitro in breast cancer cells, wherein exogenous addition of miR-34 alters cell survival post-radiation. These observations confirm that mir-34 is required for a normal cellular response to DNA damage in vivo resulting in altered cellular survival post-irradiation, and point to a potential therapeutic use for anti-miR-34 as a radiosensitizing agent in p53-mutant breast cancer.
Ovarian cancer is a major cause of cancer deaths, yet there have been few known genetic risk factors identied, the best known of which are disruptions in protein coding sequences (BRCA1 and 2). ...Recent ndings indicate that there are powerful genetic markers of cancer risk outside of these regions, in the noncoding mRNA control regions. To identify additional cancer-associated, functional non-protein-coding sequence germline variants associated with ovarian cancer risk, we captured DNA regions corresponding to all validated human microRNAs and the 3 untranslated regions (UTRs) of ~ 6000 cancer-associated genes from 31 ovarian cancer patients. Multiple single-nucleotide polymorphisms in the 3UTR of the vascular endothelial growth factor receptor/FLT1, E2F2 and PCM1 oncogenes were highly enriched in ovarian cancer patients compared with the 1000 Genome Project. Sequenom validation in a casecontrol study (267 cases and 89 controls) conrmed a novel variant in the PCM1 3UTR is significantly associated with ovarian cancer (P = 0.0086). This work identifies a potential new ovarian cancer locus and further confirms that cancer resequencing efforts should not ignore the study of noncoding regions of cancer patients.
A germline mutation in the 3′-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various ...cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).
We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings.
KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04).
The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients.
NCT00503997, NCT00425750, NCT00003809.
We have developed a tissue model of radiation-induced reproductive cell death in the nematode Caenorhabditis elegans. Reproductive cell death is the primary mode of death in tissue multipotential ...precursor cells, or "clonogens," the targets of cytotoxic therapy, whose elimination results in normal tissue damage as well as solid-tumor eradication. Through extensive morphologic and genetic analysis, we have confirmed that cell death in this model represents reproductive cell death in isolation from apoptotic cell death, affording the opportunity to define the genetic pathways required for protection from reproductive cell death. We have additionally found that the DNA damage response pathway is necessary for protection from reproductive cell death, supporting the long-held tenet that DNA damage is the cause of reproductive cell death and further validating this model. This genetic tissue model provides a valuable tool for oncology-based research and affords a platform to broaden our insight into responses to cytotoxic therapy in tissues.
MicroRNAs (miRNAs) are small ∼22nt single stranded RNAs that negatively regulate protein expression by binding to partially complementary sequences in the 3' untranslated region (3' UTRs) of target ...gene messenger RNAs (mRNA). Recently, mutations have been identified in both miRNAs and target genes that disrupt regulatory relationships, contribute to oncogenesis and serve as biomarkers for cancer risk. KIT, an established oncogene with a multifaceted role in melanogenesis and melanoma pathogenesis, has recently been shown to be upregulated in some melanomas, and is also a target of the miRNA miR-221. Here, we describe a genetic variant in the 3' UTR of the KIT oncogene that correlates with a greater than fourfold increased risk of acral melanoma. This KIT variant results in a mismatch in the seed region of a miR-221 complementary site and reporter data suggests that this mismatch can result in increased expression of the KIT oncogene. Consistent with the hypothesis that this is a functional variant, KIT mRNA and protein levels are both increased in the majority of samples harboring the KIT variant. This work identifies a novel genetic marker for increased heritable risk of melanoma.
Germline variants in the 3' untranslated region (3'UTR) of cancer genes disrupting microRNA (miRNA) regulation have recently been associated with cancer risk. A variant in the 3'UTR of the KRAS ...oncogene, referred to as the KRAS variant, is associated with both cancer risk and altered tumor biology. Here, we test the hypothesis that the KRAS variant can act as a biomarker of outcome in epithelial ovarian cancer (EOC), and investigate the cause of altered outcome in KRAS variant-positive EOC patients. As this variant seems to be associated with tumor biology, we additionally test the hypothesis that this variant can be directly targeted to impact cell survival. EOC patients with complete clinical data were genotyped for the KRAS variant and analyzed for outcome (n=536), response to neoadjuvant chemotherapy (n=125) and platinum resistance (n=306). Outcome was separately analyzed for women with known BRCA mutations (n=79). Gene expression was analyzed on a subset of tumors with available tissue. Cell lines were used to confirm altered sensitivity to chemotherapy associated with the KRAS variant. Finally, the KRAS variant was directly targeted through small-interfering RNA/miRNA oligonucleotides in cell lines and survival was measured. Postmenopausal EOC patients with the KRAS variant were significantly more likely to die of ovarian cancer by multivariate analysis (hazard ratio=1.67, 95% confidence interval: 1.09-2.57, P=0.019, n=279). Perhaps explaining this finding, EOC patients with the KRAS variant were significantly more likely to be platinum resistant (odds ratio=3.18, confidence interval: 1.31-7.72, P=0.0106, n=291). In addition, direct targeting of the KRAS variant led to a significant reduction in EOC cell growth and survival in vitro. These findings confirm the importance of the KRAS variant in EOC, and indicate that the KRAS variant is a biomarker of poor outcome in EOC likely due to platinum resistance. In addition, this study supports the hypothesis that these tumors have continued dependence on such 3'UTR lesions, and that direct targeting may be a viable future treatment approach.
Ovarian cancer is a major cause of cancer deaths, yet there have been few known genetic risk factors identified, the best known of which are disruptions in protein coding sequences (BRCA1 and 2). ...Recent findings indicate that there are powerful genetic markers of cancer risk outside of these regions, in the noncoding mRNA control regions. To identify additional cancer-associated, functional non-protein-coding sequence germline variants associated with ovarian cancer risk, we captured DNA regions corresponding to all validated human microRNAs and the 3' untranslated regions (UTRs) of ~6000 cancer-associated genes from 31 ovarian cancer patients. Multiple single-nucleotide polymorphisms in the 3'UTR of the vascular endothelial growth factor receptor/FLT1, E2F2 and PCM1 oncogenes were highly enriched in ovarian cancer patients compared with the 1000 Genome Project. Sequenom validation in a case-control study (267 cases and 89 controls) confirmed a novel variant in the PCM1 3'UTR is significantly associated with ovarian cancer (P=0.0086). This work identifies a potential new ovarian cancer locus and further confirms that cancer resequencing efforts should not ignore the study of noncoding regions of cancer patients.
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