Hydrophobic Phenoxazines Shut Down PI3K/Akt/mTOR/P70S6/S6 Kinase Signalling Pathway and Induces Massive Apoptosis in Rhabdomyosarcoma Cells

Akt plays an important role in many types of cancers and has been identified as a therapeutic target. Several types of cancers have posed a major threat to human health. Conventional treatments suffer from limitations of side effects, poor responses and drugresistance. Phenoxazines have shown diverse biological activities and promising agents in anti-cancer, anti-viral and antibacterial therapy. In this study, we evaluated the effect of phenoxazine derivatives on rhabdomyosarcoma cells. Hydrophobic phenoxazines shut down Akt/mTOR/p70S6/S6 kinase pathway and induce apoptosis in rhabdomyosarcoma cells. There is activation of Akt pathway in rhabdomyosarcoma cell lines which have tumorigenic potential. These cell lines are sensitive to phenoxazines. The phenoxazine derivatives are compared for their ability to inhibit Akt phosphorylation in these cells. The lipophilicity of these compounds increased significantly by increasing the chain length to (-CH2)5 or (-CH2)6 from the corresponding (-CH2)3 or (-CH2)4 at N10 -position of the phenoxazine ring. The ability of various phenoxazine derivatives to inhibit Akt phosphorylation in rhabdomyosarcoma cells follows the order: N10-hexyl > N10-pentyl > N10-butyl > N10-propyl. Within the series, -Cl in C-2 position on the phenoxazine ring demonstrated a higher potency compared to phenoxazines with –H in C-2 position, suggesting that chlorine is playing a critical role on the growth inhibition.

Treatment for Viral Hepatitis as Secondary Prevention for Hepatocellular Carcinoma

Chronic infections with either hepatitis B or C virus (HBV or HCV) are among the most common risk factors for developing hepatocellular carcinoma (HCC). The hepatocarcinogenic potential of these viruses is mediated through a wide range of mechanisms, including the induction of chronic inflammation and oxidative stress and the deregulation of cellular pathways by viral proteins. Over the last decade, effective anti-viral agents have made sustained viral suppression or cure a feasible treatment objective for most chronic HBV/HCV patients. Given the tumorigenic potential of HBV/HCV, it is no surprise that obtaining sustained viral suppression or eradication proves to be effective in preventing HCC. This review summarizes the mechanisms by which HCV and HBV exert their hepatocarcinogenic activity and describes in detail the efficacy of anti-HBV and anti-HCV therapies in terms of HCC prevention. Although these treatments significantly reduce the risk for HCC in patients with chronic viral hepatitis, this risk is not eliminated. Therefore, we evaluate potential strategies to improve these outcomes further and address some of the remaining controversies.

PATH-09. THE SEARCH FOR A NOVEL SUBTYPE OF GLIOBLASTOMA AND ITS CELL-OF-ORIGIN

Glioblastoma (GBM) has been computationally classified into three molecular subtypes (i.e., classical, proneural and mesenchymal). However, these subtypes lack strong biological and clinical implications. Therefore, our group has proposed to classify GBM according to its cell of origins. We have previously shown that different cell of origins give rise to biologically and transcriptionally distinct subtypes of GBM. We termed tumors that derived from subventricular zone (SVZ) neural stem cells as type 1 tumors and that from oligodendrocytic progenitor cells (OPC) as type 2 tumors. Based on murine lineage transcriptional profiles, we have also identified corresponding human GBM (40-50% of the TCGA GBM samples) tumors with conserved lineage properties. However, a majority of the TCGA GBM tumors remains unexplained by the cell-of-origin model. This study aims to search for other distinct GBM subtypes by addressing the tumorigenic potential of a putative stem progenitor population in the murine basilar pons. By using a recently reported Nestin transgenic mouse line (Nestin- C reERT2; e G FP-H2B; h D TR, or CGD in short), we have shown that conditionally deleting the commonly mutated glioma genes, Nf1 f/f ; Tp53 f/f and Pten f/+ (NPP), in pontine GFP+ cells, give rise to tumors that histologically resembles human GBM. Further transcriptomic analysis showed that a subset of these tumors highly express lineage markers of the differentiation-committed oligodendrocytic precursors (COP). We further probed the TCGA GBM database and identified 5% of the tumors to be enriched with our CGD pontine tumor-derived signature. In summary, our results showed that CGD-NPP cells can give rise to a previously uncharacterized tumor subtype with enrichment of COP lineage markers. Therefore, we propose COP as another cell of origin for GBM and that COP-derived tumor may contribute to a novel tumor subtype of the GBM classification.

Overexpression of AGR2vH, an oncogenic AGR2 spliced transcript, potentiates tumorigenicity and proteomic alterations in cholangiocarcinoma cell

ABSTRACT The upregulation of Anterior gradient 2 (AGR2) has been observed in cholangiocarcinoma (CCA) cells, nras-mutant zebrafish and specimens derived from CCA patients. Our previous study reported AGR2 splicing into AGR2vH to facilitate CCA cell aggressiveness, while this work aims to investigate the molecular mechanisms underlying AGR2vH. Firstly, AGR2vH upregulation was demonstrated in CCA tissues derived from patients. For in vitro studies, established AGR2vH-overexpressing KKU-213A cells were found to exhibit increased proliferation and clonogenicity. In vivo tumorigenicity assessed in a mouse model represented higher tumorigenic potential in AGR2vH-overexpressing cell xenograft mice. Next, LC-MS/MS was analyzed, and indicating that AGR2vH may be associated with CCA cell proliferation via Wnt/β-catenin signaling pathway activation, which was verified by β-catenin expression and nuclear translocation. The current results provide evidence that AGR2vH upregulation promotes tumorigenicity in CCA cells linked with an alteration of CCA cell proteome.

MEN1 silencing aggravates tumorigenic potential of AR-independent prostate cancer cells through nuclear translocation and activation of JunD and β-catenin

Background Recent studies highlighted the increased frequency of AR-low or -negative prostate cancers (PCas) and the importance of AR-independent mechanisms in driving metastatic castration-resistant PCa (mCRPC) development and progression. Several previous studies have highlighted the involvement of the MEN1 gene in PCa. In the current study, we focused on its role specifically in AR-independent PCa cells. Methods Cell tumorigenic features were evaluated by proliferation assay, foci formation, colony formation in soft agar, wound healing assay and xenograft experiments in mice. Quantitative RT-PCR, Western blot and immunostaining were performed to determine the expression of different factors in human PCa lines. Different ChIP-qPCR-based assays were carried out to dissect the action of JunD and β-catenin. Results We found that MEN1 silencing in AR-independent cell lines, DU145 and PC3, resulted in an increase in anchorage independence and cell migration, accompanied by sustained MYC expression. By searching for factors known to positively regulate MYC expression and play a relevant role in PCa development and progression, we uncovered that MEN1-KD triggered the nuclear translocation of JunD and β-catenin. ChIP and 3C analyses further demonstrated that MEN1-KD led to, on the one hand, augmented binding of JunD to the MYC 5′ enhancer and increased formation of loop structure, and on the other hand, increased binding of β-catenin to the MYC promoter. Moreover, the expression of several molecular markers of EMT, including E-cadherin, BMI1, Twist1 and HIF-1α, was altered in MEN1-KD DU145 and PC3 cells. In addition, analyses using cultured cells and PC3-GFP xenografts in mice demonstrated that JunD and β-catenin are necessary for the altered tumorigenic potential triggered by MEN1 inactivation in AR-independent PCa cells. Finally, we observed a significant negative clinical correlation between MEN1 and CTNNB1 mRNA expression in primary PCa and mCRPC datasets. Conclusions Our current work highlights an unrecognized oncosuppressive role for menin specifically in AR-independent PCa cells, through the activation of JunD and β-catenin pathways.

Zinc Favors Triple-Negative Breast Cancer’s Microenvironment Modulation and Cell Plasticity

Triple-negative breast cancer (TNBC) tends to metastasize to the brain, a step that worsens the patient’s prognosis. The specific hallmarks that determine successful metastasis are motility and invasion, microenvironment modulation, plasticity, and colonization. Zinc, an essential trace element, has been shown to be involved in all of these processes. In this work, we focus our attention on the potential role of zinc during TNBC metastasis. We used MDA-MB-BrM2 (BrM2) cells, a brain metastasis model derived from the parental TNBC cell line MDA-MB-231. Our studies show that BrM2 cells had double the zinc content of MDA-MB-231 cells. Moreover, exploring different metastatic hallmarks, we found that the zinc concentration is especially important in the microenvironment modulation of brain metastatic cells, enhancing the expression of SerpinB2. Furthermore, we show that zinc promotes the tumorigenic capacity of breast cancer stem cells. In addition, by causing a disturbance in MDA-MB-231 zinc homeostasis by overexpressing the Zip4 transporter, we were able to increase tumorigenicity. Nevertheless, this strategy did not completely recapitulate the BrM2 metastatic phenotype. Altogether, our work suggests that zinc plays an important role in the transformative steps that tumoral cells take to acquire tumorigenic potential and niche specificity.

Mesenchymal Stem Cells and Cancer Stem Cells: An Overview of Tumor-Mesenchymal Stem Cell Interaction for therapeutic interventions

: Tumors are made up of different types of cancer cells that contribute to tumor heterogeneity. Among these cells, cancer stem cells (CSCs) have a significant role in the onset of cancer and development. Like other stem cells, CSCs are characterized by the capacity for differentiation and self-renewal. A specific population of CSCs is constituted by mesenchymal stem cells (MSCs) that differentiate into mesoderm-specific cells. The pro-or anti-tumorigenic potential of MSCs on the proliferation and development of tumor cells has been reported as contradictory results. Also, tumor progression is specified by the corresponding tumor cells like the tumor microenvironment. The tumor microenvironment consists of a network of reciprocal cell types such as endothelial cells, immune cells, MSCs, and fibroblasts as well as growth factors, chemokines, and cytokines. In this review, recent findings related to the tumor microenvironment and associated cell populations, homing of MSCs to tumor sites, and interaction of MSCs with tumor cells will be discussed.

Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

The induction of apoptosis upon USP7 (HAUSP) inhibition is established in cancers that contain a wild-type p53 (p53Wt) through the USP7-Mdm2-p53 axis, but no clear explanation has yet been reported for the same to occur in cancers containing mutant 53 (p53Mut) or even p53 null (p53Null) systems. Instead of this USP7-Mdm2-p53 axis USP7 also works through an alternative new pathway identified in this study. Here in this study, we observed that the magnitude of apoptosis induction in response to USP7 inhibition was remarkably similar between cancer cells showing p53Null or p53Mut and those with p53Wt. Through a proteomics-based approach, we were able to identify XIAP as a novel interacting partner for USP7. XIAP is a potent and well-characterized member of the inhibitor of apoptosis proteins (IAPs), which function through caspase inhibition. We successfully identified USP7 as a positive regulator of XIAP at post-translational but not at its transcriptional level. Using molecular modelling coupled with domain deletion studies, we show that the first three Ubl domains in association with the catalytic domain of USP7 interact with the BIR2 and the linker region between BIR2 and BIR3 domains of XIAP. Modulation of expression and catalytic activity of USP7 in multiple type of cancer cell lines showed that USP7 stabilizes XIAP through its deubiquitinase activity. We have also observed that USP7 sensitizes cells against chemotherapeutic drugs through stabilization of XIAP. Thus, USP7 promotes tumorigenesis in multiple cancers, via stabilization of XIAP that results in apoptosis inhibition in caspase dependent pathway. Moreover, we observed that combinatorial inhibition of USP7 and XIAP can induce cellular apoptosis in a higher magnitude than their individual inhibition. Additionally, our results indicates that nanoformulated P5091 and P22077 showed higher potency for killing C6 cells in comparison to normal drugs. To the best of our knowledge, this is the first report on identification and validation of XIAP, a crucial E3 ubiquitin ligase, as a novel substrate of the deubiquitinase USP7 and they together involve in empowerment of the tumorigenic potential of cancer cells.

TBC1D9: An Important Modulator of Tumorigenesis in Breast Cancer

Triple-negative breast cancer (TNBC) is a major concern among the different subtypes of breast cancer (BC) due to the lack of effective treatment. In a previous study by our group aimed at understanding the difference between TNBC and non-TNBC tumors, we identified the gene TBC1 domain family member 9 (TBC1D9), the expression of which was lower in TNBC as compared to non-TNBC tumors. In the present study, analysis of TBC1D9 expression in TNBC (n = 58) and non-TNBC (n = 25) patient tumor samples validated that TBC1D9 expression can differentiate TNBC (low) from non-TNBC (high) samples and that expression of TBC1D9 was inversely correlated with grade and proliferative index. Moreover, we found that downregulation of the TBC1D9 gene decreases the proliferation marginally in non-TNBC and was associated with increased migratory and tumorigenic potential in both TNBC and luminal BC cell lines. This increase was mediated by the upregulation of ARL8A, ARL8B, PLK1, HIF1α, STAT3, and SPP1 expression in TBC1D9 knockdown cells. Our results suggest that TBC1D9 expression might limit tumor aggressiveness and that it has a differential expression in TNBC vs. non-TNBC tumors.

A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds

CD44 is emerging as an important receptor biomarker for various cancers. Amongst these is oral cancer, where surgical resection remains an essential mode of treatment. Unfortunately, surgery is frequently associated with permanent disfigurement, malnutrition, and functional comorbidities due to the difficultly of tumour removal. Optical imaging agents that can guide tumour tissue identification represent an attractive approach to minimising the impact of surgery. Here, we report the synthesis of a water-soluble fluorescent probe, namely HA-FA-HEG-OE (compound 1), that comprises components originating from natural sources: oleic acid, ferulic acid and hyaluronic acid. Compound 1 was found to be non-toxic, displayed aggregation induced emission and accumulated intracellularly in vesicles in SCC-9 oral squamous cells. The uptake of 1 was fully reversible over time. Internalization of compound 1 occurs through receptor mediated endocytosis; uniquely mediated through the CD44 receptor. Uptake is related to tumorigenic potential, with non-tumorigenic, dysplastic DOK cells and poorly tumorigenic MCF-7 cells showing only low intracellular levels and highlighting the critical role of endocytosis in cancer progression and metastasis. Together, the recognised importance of CD44 as a cancer stem cell marker in oral cancer, and the reversible, non-toxic nature of 1, makes it a promising agent for real time intraoperative imaging.