A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein.

MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX. Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.

Efficacy and Toxicity of Panitumumab After Progression on Cetuximab and Predictive Value of MiR-31-5p in Metastatic Wild-type KRAS Colorectal Cancer Patients.

BACKGROUND: In metastatic colorectal cancer (mCRC), panitumumab is generally considered to be ineffective after the progression on cetuximab therapy. However, few studies have demonstrated that a small subset of mCRC patients may benefit from panitumumab in this setting. PATIENTS AND METHODS: In our study, wild-type KRAS mCRC patients, enrolled into the nationwide Czech registry CORECT between January 2007 and December 2012, were screened for panitumumab therapy after progression on cetuximab. RESULTS: We identified 26 mCRC in the registry with well documented progression on cetuximab in combination with irinotecan-based chemotherapy (FOLFIRI or irinotecan alone) who received panitumumab monotherapy. Partial response (PR) was achieved in 3 (11.5%) patients and stable disease (SD) in 7 (26.9%) patients after 8 weeks of therapy. Thirteen (50.0%) patients had evidence of progressive disease (PD) and in 3 (11.5%) cases response was not available. Furthermore, we confirmed that higher expression levels of newly described biomarker, miR-31-5p, in tumor are significantly associated with shorter progression-free survival (PFS) in patients treated with cetuximab (p=0.038); however, we did not observe association between miR-31-5p and response to panitumumab in mCRC patients after progression on cetuximab. CONCLUSION: It remains possible that a subset of mCRC patients may benefit from panitumumab after progression on cetuximab.

Mutational analysis of primary and metastatic colorectal cancer samples underlying the resistance to cetuximab-based therapy.

PURPOSE: Although several molecular markers predicting resistance to cetuximab- or panitumumab-based therapy of metastatic colorectal cancer were described, mutations in RAS proto-oncogenes remain the only predictors being used in daily clinical practice. However, 35%-45% of wild-type RAS patients still do not respond to this anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody-based therapy, and therefore the definition of other predictors forms an important clinical need. The aim of the present retrospective single-institutional study was to evaluate potential genes responsible for resistance to anti-EGFR therapy in relation to mutational analysis of primary versus metastatic lesions. PATIENTS AND METHODS: Twenty-four paired primary and corresponding metastatic tissue samples from eight nonresponding and four responding metastatic colorectal cancer patients treated with cetuximab-based therapy were sequenced using a next-generation sequencing panel of 26 genes involved in EGFR signaling pathway and colorectal carcinogenesis. RESULTS: Mutational status of primary tumors and metastatic lesions was highly concordant in TP53, APC, CTNNB1, KRAS, PIK3CA, PTEN, and FBXW7 genes. Metastatic samples harbor significantly more mutations than primary tumors. Potentially negative predictive value of FBXW7 mutations in relationship to anti-EGFR treatment outcomes was confirmed. Finally, new occurrences of activating KRAS mutations were identified in a group of patients initially determined as wild-type RAS by routinely used qPCR-based RAS mutational tests. All newly detected activating KRAS mutations most likely led to cetuximab treatment failure. CONCLUSION: The results of the present study suggest a need of careful consideration of previously published results of anti-EGFR-targeted therapy with regard to potentially inaccurate diagnostic tools used in the past. Based on our findings, we recommend more extensive use of next-generation sequencing testing in daily clinical practice, as it brings a significant added value in terms of validity of the diagnostic procedure.

Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer.

We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P < 0.0001). By use of CRC cell lines (HCT116+/+, HCT116-/- and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified.

Prediction of response to anti-EGFR antibody-based therapies by multigene sequencing in colorectal cancer patients.

BACKGROUND: The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (moAbs) cetuximab or panitumumab are administered to colorectal cancer (CRC) patients who harbor wild-type RAS proto-oncogenes. However, a percentage of patients do not respond to this treatment. In addition to mutations in the RAS genes, mutations in other genes, such as BRAF, PI3KCA, or PTEN, could be involved in the resistance to anti-EGFR moAb therapy. METHODS: In order to develop a comprehensive approach for the detection of mutations and to eventually identify other genes responsible for resistance to anti-EGFR moAbs, we investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab. Among these, 37 samples were responsive and 28 were resistant. RESULTS: We confirmed that mutations in EGFR-pathway genes (KRAS, NRAS, BRAF, PI3KCA) were relevant for conferring resistance to therapy and could predict response (p = 0.001). After exclusion of KRAS, NRAS, BRAF and PI3KCA combined mutations could still significantly associate to resistant phenotype (p = 0.045, by Fisher exact test). In addition, mutations in FBXW7 and SMAD4 were prevalent in cases that were non-responsive to anti-EGFR moAb. After we combined the mutations of all genes (excluding KRAS), the ability to predict response to therapy improved significantly (p = 0.002, by Fisher exact test). CONCLUSIONS: The combination of mutations at KRAS and at the five gene panel demonstrates the usefulness and feasibility of multigene sequencing to assess response to anti-EGFR moAbs. The application of parallel sequencing technology in clinical practice, in addition to its innate ability to simultaneously examine the genetic status of several cancer genes, proved to be more accurate and sensitive than the presently in use traditional approaches.

MicroRNA expression profiling identifies miR-31-5p/3p as associated with time to progression in wild-type RAS metastatic colorectal cancer treated with cetuximab.

The aim of our study was to investigate whether microRNAs (miRNAs) could serve as predictive biomarkers to anti-EGFR therapy (cetuximab, panitumumab) in patients with RAS wild-type (wt-RAS) metastatic colorectal cancer (mCRC). Historical cohort of 93 patients with mCRC (2006-2009) was included and further divided into exploratory and validation cohorts. MiRNAs expression profiling was performed on the exploratory cohort of 41 wt-KRAS mCRC patients treated with cetuximab to identify miRNAs associated with time to progression (TTP). The validation was performed on two independent cohorts: 28 patients of wt-RAS mCRC treated with cetuximab and 24 patients of wt-RAS mCRC treated with panitumumab. We identified 9 miRNAs with significantly different expression between responders and non-responders to cetuximab therapy (P ≤ 0.01). These 9 miRNAs were further evaluated in two independent cohorts of patients and miR-31-3p (P < 0.001) and miR-31-5p (P < 0.001) were successfully confirmed as strongly associated with TTP in wt-RAS mCRC patients treated with cetuximab but not panitumumab. When evaluated on the complete cohort of cetuximab patients (N = 69), miR-31-3p (HR, 5.10; 95% CI, 2.52-10.32; P < 0.001) and miR-31-5p (HR, 4.80; 95% CI, 2.50-9.24; P < 0.001) were correlated with TTP on the comparable level of significance. There was no difference in miR-31-5p/3p expression levels in RAS mutated and wild-type tumor samples. MiR-31-5p/3p are promising predictive biomarkers of cetuximab response in wt-RAS mCRC patients.

MicroRNAs targeting EGFR signalling pathway in colorectal cancer.

MicroRNAs (miRNAs) are short, 18-25-nucleotide long, non-coding single-stranded RNAs, which are capable to regulate gene expression on post-transcriptional level through binding to their target protein-encoding mRNAs. miRNAs regulate individual components of multiple oncogenic pathways. One of them is epidermal growth factor receptor (EGFR) signalling pathway that regulates cell proliferation, differentiation, migration, angiogenesis and apoptosis. All these processes are deregulated in colorectal cancer (CRC). Moreover, EGFR has been validated as the therapeutic target in CRC, and monoclonal antibodies cetuximab and panitumumab are used in the therapy of patients with metastatic CRC. Because of the extensive involvement of miRNAs in the regulation of EGFR signalling, it seems they could also serve as promising predictive biomarkers to anti-EGFR therapy. In this review, we summarize current knowledge about miRNAs targeting EGFR signalling pathway, their functioning in CRC pathogenesis and potential usage as biomarkers.

Identification and functional screening of microRNAs highly deregulated in colorectal cancer.

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.