cells Targeting the Epigenetic Non-Coding RNA MALAT1 / Wnt Signaling Axis as a Therapeutic Approach to Suppress Stemness and Metastasis in Hepatocellular Carcinoma

: Background: With recorded under-performance of current standard therapeutic strategies as highlighted by high rates of post-treatment (resection or local ablation) recurrence, resistance to chemotherapy, poor overall survival, and an increasing global incidence, hepatocellular carcinoma (HCC) constitutes a medical challenge. Accumulating evidence implicates the presence of HCC stem cells (HCC-SCs) in HCC development, drug-resistance, recurrence, and progression. Therefore, treatment strategies targeting both HCC-SCs and non-CSCs are essential. Methods: Recently, there has been an increasing suggestion of MALAT1 oncogenic activity in HCC; however, its role in HCC stemness remains unexplored. Herein, we investigated the probable role of MALAT1 in the SCs-like phenotype of HCC and explored likely molecular mechanisms by which MALAT1 modulates HCC-SCs-like and metastatic phenotypes. Results: We showed that relative to normal, cirrhotic, or dysplastic liver conditions, MALAT1 was aberrantly expressed in HCC, similar to its overexpression in Huh7, Mahlavu, and SK-Hep1 HCC cells lines, compared to the normal liver cell line THLE-2. We also demonstrated a positive correlation between MALAT1 expression and poor cell di ﬀ erentiation status in HCC using RNAscope. Interestingly, we demonstrated that shRNA-mediated silencing of MALAT1 concomitantly downregulated the expression levels of β -catenin, Stat3, c-Myc, CK19, vimentin, and Twist1 proteins, inhibited HCC oncogenicity, and signiﬁcantly suppressed the HCC-SCs-related dye-e ﬄ uxing potential of HCC cells and reduced their ALDH-1 activity, partially due to inhibited MALAT1- β -catenin interaction. Additionally, using TOP / FOP (TCL / LEF-Fireﬂy luciferase) Flash, RT-PCR, and western blot assays, we showed that silencing MALAT1 downregulates β -catenin expression, dysregulates the canonical Wnt signaling pathway, and consequently attenuates HCC tumorsphere formation e ﬃ ciency, with concurrent reduction in CD133 + and CD90 + HCC cell population, and inhibits tumor growth in SK-Hep1-bearing mice. Conclusions: Taken together, our data indicate that MALAT1 / Wnt is a targetable molecular candidate, and the therapeutic targeting of MALAT1 / Wnt may constitute a novel promising anticancer strategy for HCC treatment. and adjacent non-tumor tissue samples (n = 8 pairs) from the Hospital patients cohort (n = 72) using the quantitative PCR demonstrate that the expression of MALAT1 is enhanced in most (~75%) HCC samples compared to their non-tumor counterpart, with a mean expression which is 2.66-fold higher in the HCC in comparison to the non-tumor group ( p < 0.01) (Figure 1C). These results indicate that increased MALAT1 expression is characteristic of ﬁbroblastoid, highly malignant HCC cells and tissues, and suggest its involvement in the poor cellular di ﬀ erentiation status of HCC and its associated aggressive phenotype. pool, inhibits the constitutive stemness of selected CD133 high CD90 high HCC cells, impairs the nuclear translocation of β -catenin, quells the aberrant activation of the Wnt / β -catenin, represses cisplatin-induced HCC-SCs enrichment, abrogates cancerous liver cell metastasis and clonogenicity, as well as suppresses in vivo tumor initiation and growth.