Breast cancer is a multifactorial disease and driven by aberrant regulation of cell signaling pathways due to the acquisition of genetic and epigenetic changes. An array of growth factors and their receptors is involved in cancer development and metastasis. Receptor Tyrosine Kinases (RTKs) constitute a class of receptors that play important role in cancer progression. RTKs are cell surface receptors with specialized structural and biological features which respond to environmental cues by initiating appropriate signaling cascades in tumor cells. RTKs are known to regulate various downstream signaling pathways such as MAPK, PI3K/Akt and JAK/STAT. These pathways have a pivotal role in the regulation of cancer stemness, angiogenesis and metastasis. These pathways are also imperative for a reciprocal interaction of tumor and stromal cells. Multi-faceted role of RTKs renders them amenable to therapy in breast cancer. However, structural mutations, gene amplification and alternate pathway activation pose challenges to anti-RTK therapy.

Background: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine. Main body: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo. Conclusion: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log₂ fold change ≤−1.5, P_adj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3′UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

Cancer stem cells (CSCs) are known to potentiate tumor initiation and maintenance in Oral Squamous Cell Carcinoma (OSCC). Increasing evidences suggest that CD44 ^high CD24 ^low population in OSCC are potential CSCs. MicroRNAs (miRNAs) have emerged as crucial players in tumor development. However, their role in maintenance of OSCC stem cells remains unclear. Here we report that CD44 ^high CD24 ^low population within OSCC cells and primary HNSCC tumors have an elevated expression of miR-146a. Moreover, over-expression of miR-146a results in enhanced stemness phenotype by augmenting CD44 ^high CD24 ^low population. We demonstrate that miR-146a induces stemness by stabilizing β-catenin with concomitant loss of E-cadherin and CD24. Interestingly, CD24 is identified as a novel functional target of miR-146a and ectopic expression of CD24 abrogates miR-146a driven potential CSC phenotype. Mechanistic analysis reveals that higher CD24 levels inhibit AKT phosphorylation leading to β-catenin degradation. Using stably expressing miR-146a/CD24 OSCC cell lines, we also validate that the miR-146a/ CD24/AKT loop significantly alters tumorigenic ability in vivo . Furthermore, we confirmed that β-catenin trans-activates miR-146a, thereby forming a positive feedback loop contributing to stem cell maintenance. Collectively, our study demonstrates that miR-146a regulate CSCs in OSCC through CD24-AKT-β-catenin axis. Highlights: MiR-146a induces cancer stem cell characteristics in OSCC by targeting CD24 CD24 abrogates miR-146a mediated stemness via β-catenin degradation in non-CSCs Akt/Wnt pathway is critical for sustenance of miR-146a driven potential CSCs The miR-146a/CD24/AKT loop significantly alters tumorigenic ability in vivo

Diverse mechanisms have been established to understand chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs are not surveyed well. We aimed here to explore the lncRNA/miRNA axis in hepatitis C and B virus infected HCC to investigate the mechanism of chemoresistance and to classify a potential therapeutic target for HCC. The small RNA transcriptome, and qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p/miR-136-3p/miR-139-5p/miR-223-3p/miR-375-3p were the most downregulated five miRNAs in HCC compared to normal (log₂fold change ≤-1.5, P_adj≤0.05).In-silicopathway analysis with the validated targets of each miRNA depicted that the signalling pathways regulating pluripotency of stem cells commonly targeted by the all five miRNAs. Subsequent validation by 3’UTR-luciferase assays and western blot analysis disclosed that these five miRNAs impeded either same or diverse genes, but all from BMP signaling pathways including BMPR1A/BMPR1B by miR-139-5p, miR-136-5p & miR-375-3p and ACVR2A/ACVR2B by miR424-5p & miR223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated EMT/stemness/sphere formation/chemoresistance/invasion/migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” prediction tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all five miRNAs. Ago2-RNA-immuno-precipitation/in vitro RNA pull-down assays revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered all the five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was deleted in Huh7/SNU449 cells using CRISPR-technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis of cells. Shrinkage of tumor size/volume in NOD-SCID mice injected with KCNQ1OT1-K/O cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumor milieu and modulates BMP signaling to promote chemoresistance to HCC suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target for HCC.