Anchorage-independent Growth Research Articles

TMEM52B-derived peptides inhibit generation of soluble E-cadherin and EGFR activity to suppress colon cancer growth and early metastasis

BackgroundTransmembrane protein 52B (TMEM52B) is a novel gene expressed widely in various normal human tissues; however, the biological function of TMEM52B in cancer remains largely unknown. Previously, we demonstrated that TMEM52B is a novel modulator of E-cadherin and EGFR activity, and that it has tumor suppressor-like activity using both experimental and clinical analyses. Here, we hypothesized that the extracellular domain (ECD) of TMEM52B may exert tumor-suppressing activity.MethodsWe designed and evaluated the therapeutic potential of TMEM52B ECD-derived peptides in vitro and in vivo. The molecular mechanisms underlying the anti-cancer activity of the peptides were explored.ResultsTMEM52B ECD-derived peptides reduced cancer cell survival, invasion, and anchorage-independent growth, which was accompanied by decreased phosphorylation of ERK1/2 and AKT. The peptides maintained intact E-cadherin at organized cell–cell junctions, leading to reduced β-catenin activity. They also inhibited generation of soluble E-cadherin and activation of EGFR by binding directly to the E-cadherin ECD and interfering with the interaction between soluble E-cadherin and EGFR. Peptides fused to the Fc domain of human IgG1 efficiently inhibited tumor growth in a colon cancer xenograft model and reduced survival of circulating tumor cells in an early metastasis model.ConclusionsThese results strongly suggest that TMEM52B ECD-derived peptides could provide a platform for the development of novel anti-cancer therapeutics and furnish a useful tool for exploring the function of TMEM52B in modulating the interplay between E-cadherin and EGFR.

Tumor Regulatory Effect of 15-Hydroxyprostaglandin Dehydrogenase (HPGD) in Triple-Negative Breast Cancer

Prostaglandin regulation is known to play a pivotal role in tumorigenesis; however, the contributions of the prostaglandin-metabolizing enzyme 15-hydroxyprostaglandin dehydrogenase (HPGD) to cancer development remain poorly understood. In this study, we investigate the effects of HPGD on cell viability, proliferation, anchorage-independent growth, and migration in triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer. Overexpression of HPGD in human TNBC cells resulted in both positive and negative regulation of cell proliferation and colony formation, with these effects occurring independent of prostaglandin E2 (PGE2). In contrast, overexpression of the mouse homolog, Hpgd, in murine TNBC cells led to a consistent but modest reduction in cell viability and colony formation, indicating that HPGD activity varies depending on species and cell line context. Notably, TNBC cells expressing a mutant form of Hpgd (Hpgdmut), which lacks the ability to bind PGE2, exhibited similar functional outcomes in cell viability and colony formation as those expressing wild-type Hpgd (HpgdWT). These findings suggest that HPGD may exert its tumorigenic effects through non-enzymatic mechanisms, potentially by involving modulation of KRAS signaling in human TNBC cells. Our results highlight the diverse roles of HPGD in cancer biology, particularly in the context of TNBC, and point to non-enzymatic pathways as a significant aspect of its tumorigenic activity.

Inhibition of TFF3 synergizes with c-MET inhibitors to decrease the CSC-like phenotype and metastatic burden in ER+HER2+ mammary carcinoma

The interaction between HER2 and ERα signaling pathways contributes to resistance to anti-estrogen and HER2-targeted therapies, presenting substantial treatment challenges in ER-positive (ER+) HER2-positive (HER2+) mammary carcinoma (MC). Trefoil Factor-3 (TFF3) has been reported to mediate resistance to both anti-estrogen and anti-HER2 targeted therapies in ER+ and ER+HER2+ MC, respectively. Herein, the function and mechanism of TFF3 in ER+HER2+ MC were delineated; and novel combinatorial therapeutic strategies were identified. Elevated expression of TFF3 promoted the oncogenicity of ER+HER2+ MC cells, including enhanced cell proliferation, survival, anchorage-independent growth, 3D growth, cancer stem cell-like (CSC-like) phenotype, migration, invasion, and xenograft growth. Targeting TFF3 with an interfering RNA plasmid or a small-molecule inhibitor (AMPC) inhibited these oncogenic characteristics, highlighting the therapeutic potential of targeting TFF3 in ER+HER2+ MC. Furthermore, a high-throughput combinatorial anti-cancer compound library screening revealed that AMPC preferentially synergized with receptor tyrosine kinase c-MET inhibitors (c-METis) to reduce cell survival and the CSC-like phenotype. The combination of AMPC and c-METis also synergistically suppressed the in vivo growth of ER+HER2+ MC cell-derived xenografts and abrogated lung metastasis. Mechanistically, TFF3 was observed to activate c-MET signaling through a positive-feedback loop to enhance the CSC-like phenotype of ER+HER2+ MC. Therefore, proof of concept is provided herein that antagonizing of TFF3 is a promising therapeutic strategy in combination with c-MET inhibition for the treatment of ER+HER2+ MC.

Differential Expression of miRNAs Between Young-Onset and Late-Onset Indian Colorectal Carcinoma Patients.

Reports indicate a worldwide increase in the incidence of Early-Onset Colorectal Carcinoma (EOCRC) (<50 years old). In an effort to understand the different modes of pathogenesis in early-onset CRC, colorectal tumors from EOCRC (<50 years old) and Late-Onset patients (LOCRC; >50 years old) were screened to eliminate microsatellite instability (MSI), nuclear β-catenin, and APC mutations, as these are known canonical factors in CRC pathogenesis. Small-RNA sequencing followed by comparative analysis revealed differential expression of 23 miRNAs (microRNAs) specific to EOCRC and 11 miRNAs specific to LOCRC. We validated the top 10 EOCRC DEMs in TCGA-COAD and TCGA-READ cohorts, followed by validation in additional EOCRC and LOCRC cohorts. Our integrated analysis revealed upregulation of hsa-miR-1247-3p and hsa-miR-148a-3p and downregulation of hsa-miR-326 between the two subsets. Experimentally validated targets of the above miRNAs were compared with differentially expressed genes in the TCGA dataset to identify targets with physiological significance in EOCRC development. Our analysis revealed metabolic reprogramming, downregulation of anoikis-regulating pathways, and changes in tissue morphogenesis, potentially leading to anchorage-independent growth and progression of epithelial-mesenchymal transition (EMT). Upregulated targets include proteins present in the basal part of intestinal epithelial cells and genes whose expression is known to correlate with invasion and poor prognosis.

The tumor suppressor role and epigenetic regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in cancer and tumor microenvironment (TME).

The tumor suppressor role and epigenetic regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in cancer and tumor microenvironment (TME).

Implication of the novel microtubule targeting agent, AUS_001, in pancreatic cancer cellular signaling.

748 Background: Pancreatic cancer (PanCa) is considered a major therapeutic challenge due to its poor prognosis, high mortality rate and resistance to most therapies, underlining the need for new treatment approaches. Our previous investigations revealed that the prenylated hydroxy-stilbene, AUS_001, inhibits β-tubulin polymerization via its unique binding to the colchicine site of tubulin. A cell-based profiling platform identified sensitivity of 12 out of 13 PanCa lines to AUS_001 with a median concentration causing 50% growth inhibition of 0.227µM. The goal of the current study was to elucidate the signaling activity and cellular responses following treatment with AUS_001 and explore the prospect of the latter in PanCa treatment. Methods: A series of functional and biochemical assays were performed to delineate the effect of AUS_001 on the growth, colony formation ability, invasive capacity and oxidative status of PanCa cells. Tumor growth was evaluated in PANC-1 implanted CB.17 SCID mice and MIA PaCa-2 implanted nude mice upon intraperitoneal administration of AUS_001 once weekly. Metabolic profiling was carried out using the Seahorse XF Analyzer. RNA silencing and adenovirus-mediated overexpression of the Dominant-Negative Mutant of c-Jun were employed to explore the link of c-Jun activation and AUS_001-induced cytotoxicity. Results: The potential of AUS_001 to disrupt PanCa cell functions was established in vitro and in vivo . Viability and compromised membrane integrity were multiplexed and resulted in half maximal effective concentration agreement indicating drug-induced cytotoxicity. AUS_001-treated cells underwent apoptosis followed by secondary necrosis, exerted diminished anchorage-independent growth and metastatic potential in vitro . Importantly, tumor growth was significantly reduced in PanCa murine xenograft models by AUS_001 as a monotherapy. Pretreatment with N-acetyl-l-cysteine did not alter cellular sensitivity to AUS_001, suggesting that the time-dependent induction of reactive oxygen species upon AUS_001 exposure is not the primary cause of cytotoxicity. Notably, the oxygen consumption rate, extracellular acidification rate and function of all mitochondrial complexes (I-IV) of live MIA PaCa_2 cells were significantly impaired in both glucose- and galactose-containing media 48h after AUS_001 administration. Integration of transcriptomic and proteomic data originating from AUS_001-challenged cells uncovered the dramatic induction of c-Jun and FosB stress responders. Further studies demonstrated that c-Jun transcriptional activation acts as a partial mediator of AUS_001-induced cytotoxicity. Conclusions: Collectively, our findings provide critical insights into the molecular events triggered by AUS_001 in PanCa cells and serve as supporting data for future exploration of AUS_001 as a novel PanCa therapeutic agent.

Anticancer Properties Against Select Cancer Cell Lines and Metabolomics Analysis of Tender Coconut Water.

Tender Coconut Water (TCW) is a nutrient-rich dietary supplement that contains in bioactive secondary metabolites and phytohormones with anti-oxidative and anti-inflammatory properties. Studies on TCW's anti-cancer properties are limited and the mechanism of its anti-cancer effects have not been defined. In the present study, we investigate TCW for its anti-cancer properties and, using untargeted metabolomics, we identify components form TCW with potential anti-cancer activity. Cell viability assay, BrdU incorporation assay, soft-agar assay, flow-cytometery, and Western blotting were used to analyze TCW's anticancer properties and to identify mechanism of action. Liquid chromatography- Tandem Mass Spectroscopy (LC-MS/MS) was used to identify TCW components. TCW decreased the viability and anchorage-independent growth of HepG2 hepatocellular carcinoma (HCC) cells and caused S-phase cell cycle arrest. TCW inhibited AKT and ERK phosphorylation leading to reduced ZEB1 protein, increased E-cadherin, and reduced N-cadherin protein expression in HepG2 cells, thus reversing the 'epithelial-to-mesenchymal' (EMT) transition. TCW also decreased the viability of Hep3B hepatoma, HCT-15 colon, MCF-7 and T47D luminal A breast cancer (BC) and MDA-MB-231 and MDA-MB-468 triplenegative BC cells. Importantly, TCW did not inhibit the viability of MCF-10A normal breast epithelial cells. Untargeted metabolomics analysis of TCW identified 271 metabolites, primarily lipids and lipid-like molecules, phenylpropanoids and polyketides, and organic oxygen compounds. We demonstrate that three components from TCW: 3-hydroxy-1-(4-hydroxyphenyl)propan-1-one, iondole-3-carbox aldehyde and caffeic acid inhibit the growth of cancer cells. TCW and its components exhibit anti-cancer effects. TCW inhibits the viability of HepG2 hepatocellular carcinoma cells by reversing the EMT process through inhibition of AKT and ERK signalling.

PI3K regulates TAZ/YAP and mTORC1 axes that can be synergistically targeted.

Sarcomas are a heterogeneous group of cancers with few shared therapeutic targets. PI3K signaling is activated in various subsets of sarcomas, representing a shared oncogenic signaling pathway. Oncogenic PI3K signaling has been challenging to target therapeutically. An integrated view of PI3K and Hippo pathway signaling is examined to determine if this could be leveraged therapeutically. A tissue microarray containing sarcomas of various histological types was evaluated for PTEN loss and correlated with levels of activated TAZ and YAP. PI3K and Hippo pathways were dissected in sarcoma cell lines. The role of TAZ and YAP were evaluated in a PI3K-driven mouse model. The efficacy of mTORC1 inhibition and TEAD inhibition were evaluated in sarcoma cell lines and in vivo . PI3K signaling is frequently activated in sarcomas due to PTEN loss (in 30-60%), representing a common therapeutic target. TAZ and YAP are transcriptional co-activators regulated by PI3K and drive a transcriptome necessary for tumor growth in a PI3K-driven sarcoma mouse model. Combination therapy using IK-930 (TEAD inhibitor) and everolimus (mTORC1 inhibitor) synergistically diminished proliferation and anchorage independent growth of PI3K-activated sarcoma cell lines at low, physiologically achievable doses. Furthermore, this combination therapy showed a synergistic effect in vivo , reducing tumor proliferation and size. TAZ and YAP are transcriptional co-activators downstream of PI3K signaling, a pathway that has lacked a well-defined oncogenic transcription factor. This PI3K-TAZ/YAP axis exists in parallel to the known PI3K-Akt-mTORC1 axis allowing for synergistic combination therapy targeting the TAZ/YAP-TEAD interaction and mTORC1 in sarcomas.

KNTC1 introduces segmental heterogeneity to mitochondria.

Mitochondria contribute to cellular metabolism by providing a specialised milieu for energising cells by incorporating and processing the metabolites. However, heterogeneity in the mitochondria within is only partially elucidated. Mitochondria dynamically alter their morphology and functions during the life of animals, in which cells proliferate and grow. We here show that Kntc1, a highly evolutionarily conserved protein, translocates from the Golgi apparatus to linear mitochondrial segments (LMS) upon glutamine deprivation and plays an essential role in maintaining LMS. The LMS with Kntc1 localisation exhibits an increase in the membrane potential, suggesting the role of Kntc1 in functioning as a reservoir for the energy-generating potential. Suppression of Kntc1 leads to glutamine consumption and lactate production, thus impacting cellular metabolism, eventually leading to anchorage-independent growth of cells. Indeed, the KNTC1 variant was identified in a patient with ovarian cancer, suggesting that segmental regulation of the mitochondrial function is essential for maintaining tissue integrity.

HGS Promotes Tumor Growth, Whereas the Coiled-Coil Domain and Its Oligopeptide of HGS Suppress It.

We previously isolated a cDNA clone for galactosylceramide expression factor 1, which is the rat homologue of hepatocyte-growth-factor-regulated tyrosine kinase substrate (HGS) and induces galactosylceramide expression and morphological changes in COS-7 cells, and reported that overexpression of HGS induced morphological changes in canine kidney epithelial MDCK cells. HGS is a component of the endosomal sorting complexes required for transport machinery that mediates endosomal multivesicle body formation. In this study, the overexpression of HGS induced epithelial-mesenchymal transition and caused transformation in MDCK cells, whereas the overexpression of a coiled-coil domain of HGS inhibited induction of epithelial-mesenchymal transition by HGF stimulation. The overexpression of HGS in mouse melanoma B16 cells and human colorectal cancer COLO205 cells promoted cancer characteristic anchorage-independent cell growth ability and tumor growth, whereas the overexpression of the coiled-coil domain of HGS in these cells suppressed them. The oligopeptide OP12-462 constituting the coiled-coil domain suppressed the anchorage-independent cell growth ability and tumor growth of COLO205 cells. The coiled-coil domain of HGS and OP12-462 are novel tumor growth inhibitors that do not directly destroy cancer cells but rather inhibit only the anchorage-independent cell growth ability of cancer cells.

Vitamin K-dependent gamma-carboxyglutamic acid protein 1 promotes pancreatic ductal adenocarcinoma progression through stabilizing oncoprotein KRAS and tyrosine kinase receptor EGFR.

Vitamin K-dependent γ-glutamic acid carboxylation (Gla) proteins are calcium-binding and membrane-associated, participating in coagulation, bone turnover, and cancer biology. The molecular function of transmembrane proline-rich Gla proteins (PRRGs) remains unexplored. Analysis of pancreatic ductal adenocarcinoma (PDAC) datasets, including transcription profiles, clinical data, and tissue microarrays, was conducted to evaluate PRRG1 expression and its clinical relevance. PDAC cell lines with overexpressed, knockdown, and mutated PRRG1 were developed to study biological functions and pathways using RNA-seq, co-immunoprecipitation with mass spectrometry, Western blotting, and immunofluorescence. In vivo xenograft and orthotopic models assessed PRRG1's impact on PDAC progression, with and without warfarin treatment. PRRG1 was significantly upregulated in PDAC compared to normal pancreas, correlating with poorer patient survival. PRRG1 knockdown reduced PDAC cell proliferation, anchorage-independent growth in vitro, and tumor growth in vivo. PRRG1 localized at the plasma membrane, interacted with the HECT E3 ligase NEDD4 via the C-terminal PPXY motif, and promoted NEDD4 self-ubiquitination, reducing its protein levels. PRRG1 knockdown elevated NEDD4, destabilizing the oncoprotein KRAS and receptor EGFR, and attenuating downstream signaling and macropinocytosis under nutrient deprivation. The vitamin K-dependent Gla modification of PRRG1 was crucial for its membrane localization and pro-tumorigenic effects, and was inhibited by low-dose warfarin, a clinical vitamin K antagonist. This study identifies PRRG1 as a key regulator of pro-tumorigenic signaling in PDAC, suggesting the potential of repurposing the anticoagulant warfarin as a therapeutic strategy. PRRG1 is identified as the transmembrane Gla protein mediating PDAC malignancy. PRRG1 recruits and induces self-ubiquitination of membrane-anchoring E3 ligase NEDD4. PRRG1 exerts a protective role toward KRAS and EGFR by inhibiting NEDD4. The anticoagulant warfarin can be utilized to inhibit PRRG1 and PDAC advancement.

Identification of Translocon-associated Protein Delta as An Oncogene in Human Colorectal Cancer Cells.

Identifying the roles of genes in cancer is critical in discovering potential genetic therapies for cancer care. Translocon-associated protein delta (TRAPδ), also known as signal sequence receptor 4 (SSR4), is a constituent unit in the TRAP/SSR complex that resides in the endoplasmic reticulum and plays a key role in transporting newly synthesized proteins into the endoplasmic reticulumn. However, its biological role in disease development remains unknown to date. This is the first study to identify the role of TRAPδ/SSR4 in colorectal cancer cells in vitro. Upon successful transient knockdown of TRAPδ/SSR4, we observed significant reduction of cell viability in all colorectal cancer cell lines tested. Both HCT 116 and SW480 cell lines were significantly arrested at S and G1 phases, while DLD-1 cells were significantly apoptotic. Moreover, TRAPδ/SSR4 stable knockdown HCT 116 and SW480 cells showed significantly lower viability, anchorage-independent growth, and increased S and G1 phase arrests. Overall, we conclude TRAPδ/SSR4 is a potential oncogene in human colorectal cancer cells.

The efficacy of an embryonic stem cell-based vaccine for lung cancer prevention depends on the undifferentiated state of the stem cells

Based on the antigenic similarity between tumor cells and embryonic stem cells (ESCs), several recent studies report the use of intact murine ESCs or exosomes from murine ESCs as cancer vaccines. Since the capacity for self-renewal is one of the most specialized properties shared between ESCs and a subset of tumor cells, cancer stem cells (CSCs), we investigated whether the undifferentiated state of murine ESCs is essential for the prophylactic effectiveness of an ESC-based vaccine. The undifferentiated state of ES-D3, a murine ESC line, was essential for their anchorage-independent growth potential. Importantly, differentiation of ES-D3 cells decreased their efficacy in preventing the outgrowth of implanted lung tumors. Furthermore, the long-term cancer-preventive potential of this vaccine was also inhibited by the differentiation of these cells. To examine the antigenicity of the ESC-derived vaccine, we performed combined affinity chromatography shotgun immunoproteomic experiments to identify antigens specific to the whole-cell ES vaccine as well as to the ESC-derived exosome vaccine. Our data demonstrate that antibodies against several lung cancer-associated keratin members were enriched in the serum of vaccinated mice. In summary, these data suggest that the tumor-preventing efficacy of ESC-based vaccine is reliant on the differentiation properties of these stem cells.

Epidermal growth factor receptor ligands enriched in follicular fluid exosomes promote oncogenesis of fallopian tube epithelial cells

BackgroundIncessant ovulation is the main etiologic factor of ovarian high-grade serous carcinomas (HGSC), which mostly originate from the fallopian tube epithelium (FTE). Receptor tyrosine kinase (RTK) ligands essential for follicle development and ovulation wound repair were abundant in the follicular fluid (FF) and promoted the transformation of FTE cells. This study determined whether RTK ligands are present in FF exosomes and whether epidermal growth factor receptor (EGFR) signaling is essential for oncogenic activity.MethodsThe FF of women undergoing in vitro fertilization was fractionated based on the richness of exosomes and tested for transformation toward FTE cells under different RTK inhibitors. EGFR ligands in FF exosomes were identified, and downstream signaling proteins in FTE cells were characterized.ResultsThe transforming activity of FF was almost exclusively enriched in exosomes, which possess a high capacity to induce anchorage-independent growth, clonogenicity, migration, invasion, and proliferation of FTE cells. EGFR inhibition abolished most of these activities. FF and FF exosome exposure markedly increased EGFR phosphorylation and the downstream signal proteins, including AKT, MAPK, and FAK. Multiple EGF family growth factors, such as amphiregulin, epiregulin, betacellulin, and transforming growth factor-alpha, were identified in FF exosomes.ConclusionsOur results demonstrate that FF exosomes serve as carriers of EGFR ligands as well as ligands of other RTKs that mediate the transformation of FTE cells and underscore the need to further explore the content and roles of FF exosomes in HGSC development.

EFFECT OF ADHESIVE LLC CELL PRETREATMENT BY OXAMATE ON THE SURVIVAL INDEXES AFTER TRANSITION TO DE-ADHESIVE GROWTH.

The ability to metabolic reprogramming is a distinctive feature of metastatically active tumor cells. A classic example of metabolic reprogramming, characteristic of almost all malignant cells, is aerobic glycolysis. Therefore, inhibition of glycolysis in tumor cells is considered a promising strategy for antitumor therapy. To generate Lewis lung carcinoma (LLC) cell subpopulation after pretreatment by a lactate dehydrogenase (LDH) inhibitor - oxamate in adhesive growth conditions, and then to study the metabolism of this subpopulation in the anchorage-independent growth conditions. LLC cells were cultured without oxamate or with 17 mM oxamate in the adhesive growth conditions with the following transition to the anchorage-independent growth conditions without oxamate. A distribution of LLC cells by cell cycle phases, apoptosis rate, levels of reactive oxygen species (ROS), E-cadherin, and vimentin were determined by flow cytometry. Glucose consumption and lactate production were determined by spectrophotometry. 48-h oxamate treatment in adhesive growth conditions resulted in a 30% decrease of the total number of LLC cells compared to the control. In 72 h after the transfer of both oxamate-treated and control cells into the anchorage-independent growth condition without oxamate, the number of viable cells pretreated with oxamate was reduced by 17% (p < 0.05) compared to the control cells. However, the distribution of cells by cell cycle phases did not differ. In cells pre-treated with oxamate, the rate of glucose consumption decreased by 20% (p < 0.05), ROS generation was reduced by 17%, vimentin expression decreased by 10% while the rate of lactate production was the same in oxamate-pretreated and control cells. The cytostatic effect of oxamate demonstrated in adhesive growth conditions persisted for 72 h in the anchorage-independent growth conditions. The absence of differences in the cell cycle phase distribution and a decrease in the ROS generation may indicate the initial stage of overcoming the cytostatic effect of oxamate after 72 h of culturing LLC cells in anchorage- independent growth conditions.

Establishment of two novel organoid lines from patients with combined hepatocellular cholangiocarcinoma.

Combined hepatocellular cholangiocarcinoma (cHCC-CCA) is a unique subtype of primary liver cancer displaying both hepatocytic and cholangiocytic differentiation. The development of effective treatments for cHCC-CCA remains challenging because of its high heterogeneity and lack of a suitable model system. Using a three-dimensional culture system, we successfully established two novel cHCC-CCA organoid lines from patients undergoing surgical resection for primary liver cancer. cHCC-CCA organoid lines were authenticated by fingerprint analysis, and their morphology, growth kinetics, and anchorage-independent growth were also characterized. Hematoxylin and eosin staining and immunohistochemical analysis showed that the cHCC-CCA organoids preserved the growth pattern, differentiation grade, and phenotypic characteristics of their parental tumors. Whole-exome sequencing demonstrated that patient-derived cHCC-CCA organoid lines retained the genetic alterations identified in their original tumors. Subcutaneous tumors developed in immunodeficient mice after injection of cHCC-CCA organoids. Histologically, the xenografts recapitulated the features of the original cHCC-CCA tumors, harboring both HCC and intrahepatic cholangiocarcinoma components within the same tumor. The establishment of patient-derived cHCC-CCA organoid lines with high tumorigenicity provides a valuable resource for the mechanistic investigation and drug development of this disease.

MiR-198 targets TOPORS: implications for oral squamous cell carcinoma pathogenesis.

miRNAs play a critical role in the progression of various diseases, including oral squamous cell carcinoma (OSCC), which represents a major health concern and is one of the leading causes for new cancer cases worldwide. The miRNA dysregulation causes havoc and could be attributed to various factors, with epigenetic silencing of tumor suppressor genes being a major contributor to tumorigenesis. In this study, we have explored the tumor suppressive role of miR-198 in OSCC. The tumor suppressive effect of miR-198 is established using miRNA analysis in OSCC cell lines, patient samples and xenograft nude mice model. The relationship between the miR-198 and TOPORS is explored using bioinformatics analyses, qRT-PCR, dual-luciferase reporter assay, Western blotting and cancer hall marks assays. The hypermethylation of the MIR198 promoter is confirmed using bisulfite sequencing PCR. We have found miR-198 to be upregulated in OSCC cells treated with 5-Azacytidine, a known DNA methyltransferase inhibitor. Upregulation of miR-198 in 5-Azacytidine treated OSCC cells appears to be due to methylation of the MIR198 promoter. Using bioinformatics analysis and dual-luciferase reporter assay, we have identified TOPORS (TOP1 binding arginine/serine rich protein, E3 ubiquitin ligase) as a novel gene target for miR-198. miR-198-mediated repression of TOPORS decreases cell proliferation and anchorage-independent growth and enhances apoptosis of OSCC cells, which is dependent on the presence of the 3'UTR in TOPORS. An inverse correlation between the expression levels of miR-198 and TOPORS is observed in OSCC patient samples, highlighting the biological relevance of their interaction. Delivery of a synthetic miR-198 mimic to OSCC cells results in a significant decrease in xenograft size in nude mice, potentiating its use in therapeutics. These results suggest that miR-198 is epigenetically silenced in OSCC, which promotes tumor growth, in part, by upregulating the levels of TOPORS.

Mycobacteria Treatment Inhibits Bladder Cancer Cell Migration, Invasion, and Anchorage-Independent Growth.

Bladder cancer (BC) is a highly recurrent and invasive malignancy, with Mycobacterium bovis BCG serving as the primary immunotherapy, particularly for non-muscle-invasive bladder cancer (NMIBC). However, the mechanisms underlying BCG's antitumor effects and the potential of non-tuberculous mycobacteria like Mycobacterium brumae remain unclear. This study investigates the antitumor effects of M. bovis BCG and M. brumae on BC cell migration, invasion, and anchorage-independent growth. BC cell lines representing different stages of tumor differentiation were treated with either M. bovis BCG or M. brumae. Cell migration was assessed through wound healing and transwell assays, invasiveness by transwell invasion assays, MMP-9 production by gelatin zymography, and anchorage-independent growth via soft agar colony formation. Both mycobacteria inhibited individual cell migration across all BC lines, while collective migration was only reduced in intermediate-grade cells. Both treatments also reduced invasiveness, associated with decreased MMP-9 production. Furthermore, M. brumae inhibited anchorage-independent growth across all BC lines, while M. bovis BCG had a more selective effect, primarily inhibiting growth in high-grade cells. In conclusion, both mycobacteria reduce migration, invasion, and anchorage-independent growth of BC cells, with their effectiveness varying by species and tumor differentiation grade.

Biased signaling by mutant EGFR underlies dependence on PKCα in lung adenocarcinoma

Biased signaling by mutant EGFR underlies dependence on PKCα in lung adenocarcinoma

Promoter hypermethylation-mediated downregulation of PAX6 promotes tumor growth and metastasis during the progression of liver cancer

BackgroundThe progression of liver cancer is a complicated process that involves genetic and epigenetic changes. Paired box 6 (PAX6) is a critical transcription factor for embryonic development. PAX6 is abnormally methylated in human cancer. The role of the PAX6 gene in the pathogenesis of hepatocellular carcinoma (HCC) is still unclear.MethodsTranscriptional silencing of PAX6 mediated by promoter methylation was confirmed using quantitative methylation-specific polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. Then we conducted gain-and-loss of function approaches to evaluate the function of PAX6 in HCC progression in vitro. Moreover, we designed xenograft mouse models to assess the effect of PAX6 on tumor growth and metastasis. Finally, we used RNA sequencing (RNA-seq) strategy and phenotypic rescue experiments to identify potential targets of PAX6 performing tumor-suppressive function.ResultsConstitutive expression of PAX6 suppressed anchorage-independent growth and cell invasion in vitro as well as tumor growth and metastasis in xenograft mouse models. In contrast, the inhibition of PAX6 using knockout and knockdown strategies increased tumor growth both in vitro and in vivo. Downregulation of PAX6 by doxycycline depletion partially reversed the malignant phenotypes of HCC cells induced by PAX6. Moreover, we identified E-cadherin (CDH1) and thrombospondin-1 (THBS1) as targets of PAX6. Ultimately, we demonstrated that the knockdown of CDH1 and overexpression of THBS1 in PAX6-expressing HCC cells partly reversed the tumor-suppressive effect.ConclusionPAX6 functions as a tumor suppressor partly through upregulation of CDH1 and downregulation of THBS1. Promoter hypermethylation-mediated suppression of PAX6 reduces the tumor suppressor function in the progression of liver cancer.