The Long Noncoding RNA Cancer Susceptibility 9 and RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein L Form a Complex and Coregulate Genes Linked to AKT Signaling

Abstract

The identification of viability-associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability-associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up-regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)-mediated and siRNA pool-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9-depleted and HNRNPL-depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient-derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.

AKTions by Cytoplasmic lncRNA CASC9 Promote Hepatocellular Carcinoma Survival

“…The authors identified and studied in molecular detail a prominent cytoplasmic lncRNA, CASC9 (cancer susceptibility candidate 9), which was preferentially abundant in HCC compared with normal liver. CASC9 had recently been shown to promote tumorigenesis in the esophagus, stomach, lung, and nasopharynx; in keeping with these findings, CASC9 was also significantly induced in HCCs and correlated inversely with patient survival . Moreover, a series of loss‐of‐function studies revealed that higher expression of CASC9 was strongly associated with major phenotypes observed in liver tumorigenesis, such as excessive proliferation and loss of susceptibility to apoptosis.…”

“…In this issue of Hepatology, Klingenberg et al (5) report a comprehensive collection of viability-associated lncRNAs in HCC. The authors identified and studied in molecular detail a prominent cytoplasmic lncRNA, CASC9 (cancer susceptibility candidate 9), which was preferentially abundant in HCC compared with normal liver.…”

“…CASC9 had recently been shown to promote tumorigenesis in the esophagus, stomach, lung, and nasopharynx; in keeping with these findings, CASC9 was also significantly induced in HCCs and correlated inversely with patient survival. (5) Moreover, a series of loss-of-function studies revealed that higher expression of CASC9 was strongly associated with major phenotypes observed in liver tumorigenesis, such as excessive proliferation and loss of susceptibility to apoptosis. However, in contrast to earlier studies reporting that CASC9 was primarily nuclear in other models of carcinogenesis, Klingenberg et al (5) found that CASC9 was largely cytoplasmic in HCC.…”

“…(5) Moreover, a series of loss-of-function studies revealed that higher expression of CASC9 was strongly associated with major phenotypes observed in liver tumorigenesis, such as excessive proliferation and loss of susceptibility to apoptosis. However, in contrast to earlier studies reporting that CASC9 was primarily nuclear in other models of carcinogenesis, Klingenberg et al (5) found that CASC9 was largely cytoplasmic in HCC. Interestingly, quantitative proteomic analysis using SILAC (stable isotope labeling by amino acids in cell culture) revealed that a major role of CASC9 was to activate the cytoplasmic kinase AKT, as well as the AKT-related network of signaling molecules, including upstream and downstream AKT regulators (e.g., PI3K, mTOR, PTEN, PDK1/2).…”

“…Thus, interventions to lower CASC9 to inactivate AKT would be expected to dampen cell survival and mitogenic pathways widely and robustly. The impact of CASC9 on HCC cell survival and proliferation was reflected in the broad spectrum of mRNAs and proteins differentially expressed when CASC9 was silenced . The diverse gene expression programs affected by CASC9 abundance likely reflected the fact that the downstream targets of AKT, both direct and indirect, included many transcription factors (e.g., FOXO, NFκB, TP53, HIF1A) and translational regulators (e.g., TSC1/2, mTOR).…”