Suppression of miR-199a maturation by HuR is crucial for hypoxia-induced glycolytic switch in hepatocellular carcinoma.

Glucose metabolic reprogramming is a hallmark of cancer. Cancer cells rapidly adjust their energy source from oxidative phosphorylation to glycolytic metabolism in order to efficiently proliferate in a hypoxic environment, but the mechanism underlying this switch is still incompletely understood. Here, we report that hypoxia potently induces the RNA-binding protein HuR to specifically bind primary miR-199a transcript to block miR-199a maturation in hepatocellular carcinoma (HCC) cells. We demonstrate that this hypoxia-suppressed miR-199a plays a decisive role in limiting glycolysis in HCC cells by targeting hexokinase-2 (Hk2) and pyruvate kinase-M2 (Pkm2). Furthermore, systemically delivered cholesterol-modified agomiR-199a inhibits [(18)F]-fluorodeoxyglucose uptake and attenuates tumor growth in HCC tumor-bearing mice. These data reveal a novel mechanism of reprogramming of cancer energy metabolism in which HuR suppresses miR-199a maturation to link hypoxia to the Warburg effect and suggest a promising therapeutic strategy that targets miR-199a to interrupt cancerous aerobic glycolysis.

MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFßR2.

MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3'-untranslated region (3'-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFßR2 expression by targeting one binding site in the 3'-UTR of TGFßR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFßR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFßR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFßR2.

A novel microRNA-182/Interleukin-8 regulatory axis controls osteolytic bone metastasis of lung cancer.

Bone metastasis is one of the main complications of lung cancer and most important factors that lead to poor life quality and low survival rate in lung cancer patients. However, the regulatory mechanisms underlying lung cancer bone metastasis are still poor understood. Here, we report that microRNA-182 (miR-182) plays a critical role in regulating osteoclastic metastasis of lung cancer cells. We found that miR-182 was significantly upregulated in both bone-metastatic human non-small cell lung cancer (NSCLC) cell line and tumor specimens. We further demonstrated that miR-182 markedly enhanced the ability of NSCLC cells for osteolytic bone metastasis in nude mice. Mechanistically, miR-182 promotes NSCLC cells to secrete Interleukin-8 (IL-8) and in turn facilitates osteoclastogenesis via activating STAT3 signaling in osteoclast progenitor cells. Importantly, systemically delivered IL-8 neutralizing antibody inhibits NSCLC bone metastasis in nude mice. Collectively, our findings identify the miR-182/IL-8/STAT3 axis as a key regulatory pathway in controlling lung cancer cell-induced osteolytic bone metastasis and suggest a promising therapeutic strategy that targets this regulatory axis to interrupt lung cancer bone metastasis.

Histone-Related Genes Are Hypermethylated in Lung Cancer and Hypermethylated HIST1H4F Could Serve as a Pan-Cancer Biomarker.

Lung cancer is the leading cause of cancer-related deaths worldwide. Cytologic examination is the current "gold standard" for lung cancer diagnosis, however, this has low sensitivity. Here, we identified a typical methylation signature of histone genes in lung cancer by whole-genome DNA methylation analysis, which was validated by The Cancer Genome Atlas (TCGA) lung cancer cohort (n = 907) and was further confirmed in 265 bronchoalveolar lavage fluid samples with specificity and sensitivity of 96.7% and 87.0%, respectively. More importantly, HIST1H4F was universally hypermethylated in all 17 tumor types from TCGA datasets (n = 7,344), which was further validated in nine different types of cancer (n = 243). These results demonstrate that HIST1H4F can function as a universal-cancer-only methylation (UCOM) marker, which may aid in understanding general tumorigenesis and improve screening for early cancer diagnosis. SIGNIFICANCE: These findings identify a new biomarker for cancer detection and show that hypermethylation of histone-related genes seems to persist across cancers.

Detection of Plasma EGFR Mutations in NSCLC Patients with a Validated ddPCR Lung cfDNA Assay.

Purpose: The clinical utility of cell-free DNA (cfDNA) to assess EGFR mutations is increasing. However, there are limited studies determining their clinical validity and utility. The value of cfDNA assays in cancer management remains controversial. Methods: In this study, we first evaluated the analytical performance of the ddPCR Lung cfDNA Assay. We next analyzed the concordance of the results with tissue amplification refractory mutation system PCR (ARMS-PCR) and plasma next-generation sequencing (NGS) genotyping. Finally, we assessed its clinical utility by exploring the association of cfDNA EGFR mutations with metastatic sites and the efficacy of EGFR-TKIs treatment. Results: The ddPCR Lung cfDNA Assay demonstrated a limit of blank of 1 droplet per reaction, an analytical specificity of 100%, and detection limit of 0.05%, 0.05%, and 0.1% for E746_A750del, L858R, and T790M, respectively. With tissue ARMS-PCR as a standard for comparison, the clinical sensitivity and specificity of ddPCR were 62.5% (15/24) and 100% (82/82) for E746_A750del, and 75.0% (15/20) and 94.2% (81/86) for L858R, respectively. The ddPCR showed high concordance with NGS in determining cfDNA EGFR mutations. Patients with bone and/or brain metastasis showed a higher detection rate and mutant abundance of cfDNA EGFR mutations compared to those with other sites of metastasis. Moreover, EGFR-TKIs treatment was effective in patients with sensitive EGFR mutations in either plasma cfDNA or tumor tissue-derived DNA. Conclusions: We validated in this study that the ddPCR Lung cfDNA Assay is reliable for detection of EGFR mutations in lung cancers, in terms of analytical performance, clinical validity and utility.

MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2.

Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3'-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.

IL-1ß-mediated repression of microRNA-101 is crucial for inflammation-promoted lung tumorigenesis.

Inflammatory stimuli clearly contribute to lung cancer development and progression, but the underlying pathogenic mechanisms are not fully understood. We found that the proinflammatory cytokine IL-1ß is dramatically elevated in the serum of patients with non-small cell lung cancer (NSCLC). In vitro studies showed that IL-1ß promoted the proliferation and migration of NSCLC cells. Mechanistically, IL-1ß acted through the COX2-HIF1α pathway to repress the expression of microRNA-101 (miR-101), a microRNA with an established role in tumor suppression. Lin28B was identified as critical effector target of miR-101 with its repression of Lin28B, a critical aspect of tumor suppression. Overall, IL-1ß upregulated Lin28B by downregulating miR-101. Interestingly, cyclooxygenase-2 inhibition by aspirin or celecoxib abrogated IL-1ß-mediated repression of miR-101 and IL-1ß-mediated activation of Lin28B along with their stimulatory effects on NSCLC cell proliferation and migration. Together, our findings defined an IL-1ß-miR-101-Lin28B pathway as a novel regulatory axis of pathogenic inflammatory signaling in NSCLC.