Recurrent UBE3C-LRP5 translocations in head and neck cancer with therapeutic implications.

Head and neck cancer is a major cause of morbidity and mortality worldwide. The identification of genetic alterations in head and neck cancer may improve diagnosis and treatment outcomes. In this study, we report the identification and functional characterization of UBE3C-LRP5 translocation in head and neck cancer. Our whole transcriptome sequencing and RT-PCR analysis of 151 head and neck cancer tumor samples identified the LRP5-UBE3C and UBE3C-LRP5 fusion transcripts in 5.3% of patients of Indian origin (n = 151), and UBE3C-LRP5 fusion transcripts in 1.2% of TCGA-HNSC patients (n = 502). Further, whole genome sequencing identified the breakpoint of UBE3C-LRP5 translocation. We demonstrate that UBE3C-LRP5 fusion is activating in vitro and in vivo, and promotes the proliferation, migration, and invasion of head and neck cancer cells. In contrast, depletion of UBE3C-LRP5 fusion suppresses the clonogenic, migratory, and invasive potential of the cells. The UBE3C-LRP5 fusion activates the Wnt/ß-catenin signaling by promoting nuclear accumulation of ß-catenin, leading to upregulation of Wnt/ß-catenin target genes, MYC, CCND1, TCF4, and LEF1. Consistently, treatment with the FDA-approved drug, pyrvinium pamoate, significantly reduced the transforming ability of cells expressing the fusion protein and improved survival in mice bearing tumors of fusion-overexpressing cells. Interestingly, fusion-expressing cells upon knockdown of CTNNB1, or LEF1 show reduced proliferation, clonogenic abilities, and reduced sensitivity to pyrvinium pamoate. Overall, our study suggests that the UBE3C-LRP5 fusion is a promising therapeutic target for head and neck cancer and that pyrvinium pamoate may be a potential drug candidate for treating head and neck cancer harboring this translocation.

Histone acetylation: a key determinant of acquired cisplatin resistance in cancer.

Cisplatin is an alkylating class of chemotherapeutic drugs used to treat cancer patients. However, cisplatin fails in long-term treatment, and drug resistance is the primary reason for tumor recurrence. Hence, understanding the mechanism of acquirement of chemoresistance is essential for developing novel combination therapeutic approaches. In this study, in vitro cisplatin-resistant cancer cell line models were developed. Gene ontology and GSEA of differentially expressed genes between parental and resistant cells suggest that PI3K-AKT signaling, central carbon metabolism, and epigenetic-associated phenomenon alter in cisplatin-resistant cells. Further, the data showed that increased glucose transport, alteration in the activity of histone-modifying enzymes, and acetyl-CoA levels in resistant cells paralleled an increase in global histone acetylation. Enrichment of histone acetylation on effectors of PI3K-AKT and glycolysis pathway provides evidence of epigenetic regulation of the key molecules in drug resistance. Moreover, cisplatin treatment to resistant cells showed no significant changes in histone acetylation marks since drug treatment alters cell epigenome. In continuation, targeting PI3K-AKT signaling and glycolysis leads to alteration in histone acetylation levels and re-sensitization of resistant cells to chemo-drug. The data provide evidence of histone acetylation's importance in regulating pathways and cisplatin-resistant cells' cell survival. Our study paves the way for new approaches for developing personalized therapies in affecting metabolic pathways and epigenetic changes to achieve better outcomes for targeting drug-resistant cells.

Thearubigins/Polymeric black tea polyphenols (PBPs) do not prevent benzo[a]pyrene (B[a]P) induced lung tumors in A/J mice.

OBJECTIVES: Previously we have demonstrated the chemopreventive effect of Thearubigins/Polymeric Black-tea Polyphenols (PBPs) upon pre-treatment to a combination of carcinogens, that is, Benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which are present in Tobacco smoke (TS). However, the chemopreventive effect in response to B[a]P as a single carcinogen remains unexplored. B[a]P is a universal carcinogen and an important constituent of particulate matter 2.5 (PM2.5) found in the environment and in TS. METHODS: We investigated the pre-treatment of Thearubigins/PBPs as a chemopreventive agent at three doses (1.5, 5, 10%) against B[a]P-induced lung carcinogenesis at early & late time points. We also investigated the effect of PBPs at early time points to understand molecular changes by employing western blotting in xenobiotic metabolism pathways, DNA damage, inflammation, apoptosis, and proliferation as they are modulated in response to carcinogens. We used 6-8 weeks male A/J mice for tumorigenicty and western blotting to probe the molecular biomarkers. RESULTS: We report no decrease in tumor incidence and multiplicity upon pre and concurrent treatment of Thearubigins/PBPs. Further, we also report no changes in molecular markers at early time points, in agreement with former observations. CONCLUSION: Our results suggest that chemopreventive agents need to be tested with different combinations of carcinogens and regimens to fully understand the complex interplay between carcinogenesis and the efficacy of chemopreventive agents. Studies like these will provide meaningful data before initiating large-scale clinical trials.

Role of miR-944/MMP10/AXL- axis in lymph node metastasis in tongue cancer.

Occult lymph-node metastasis is a crucial predictor of tongue cancer mortality, with an unmet need to understand the underlying mechanism. Our immunohistochemical and real-time PCR analysis of 208 tongue tumors show overexpression of Matrix Metalloproteinase, MMP10, in 86% of node-positive tongue tumors (n = 79; p < 0.00001). Additionally, global profiling for non-coding RNAs associated with node-positive tumors reveals that of the 11 significantly de-regulated miRNAs, miR-944 negatively regulates MMP10 by targeting its 3'-UTR. We demonstrate that proliferation, migration, and invasion of tongue cancer cells are suppressed by MMP10 knockdown or miR-944 overexpression. Further, we show that depletion of MMP10 prevents nodal metastases using an orthotopic tongue cancer mice model. In contrast, overexpression of MMP10 leads to opposite effects upregulating epithelial-mesenchymal-transition, mediated by a tyrosine kinase gene, AXL, to promote nodal and distant metastasis in vivo. Strikingly, AXL expression is essential and sufficient to mediate the functional consequence of MMP10 overexpression. Consistent with our findings, TCGA-HNSC data suggests overexpression of MMP10 or AXL positively correlates with poor survival of the patients. In conclusion, our results establish that the miR-944/MMP10/AXL- axis underlies lymph node metastases with potential therapeutic intervention and prediction of nodal metastases in tongue cancer patients.

Substrate stiffness regulates the recurrent glioblastoma cell morphology and aggressiveness.

Recurrent glioblastoma is highly aggressive with currently no specific treatment regime. Therefore, to identify novel therapeutic targets for recurrent GBM, we used a cellular model developed in our lab from commercially available cell line U87MG and patient-derived cultures that allows the comparison between radiation naïve (Parent) and recurrent GBM cells generated after parent cells are exposed to lethal dose of radiation. Total RNA-seq of parent and recurrent population revealed significant upregulation of cell-ECM interactions pathway in the recurrent population. These results led us to hypothesize that the physical microenvironment contributes to the aggressiveness of recurrent GBM. To verify this, we cultured parent and recurrent GBM cells on collagen-coated polyacrylamide gels mimicking the stiffness of normal brain (Young's modulus E = 0.5kPa) or tumorigenic brain (E = 10kPa) and tissue culture plastic dishes (E ∼ 1 GPa). We found that compared to parent cells, recurrent cells showed higher proliferation, invasion, migration, and resistance to EGFR inhibitor. Using orthotopic GBM mouse model and resection model, we demonstrate that recurrent cells cultured on 0.5kPa had higher in vivo tumorigenicity and recurrent disease progression than parent cells, whereas these differences were insignificant when parent and recurrent cells were cultured on plastic substrates. Furthermore, recurrent cells on 0.5kPa showed high expression of ECM proteins like Collagen, MMP2 and MMP9. These proteins were also significantly upregulated in recurrent patient biopsies. Additionally, the brain of mice injected with recurrent cells grown on 0.5kPa showed higher Young's moduli suggesting the ability of these cells to make the surrounding ECM stiffer. Total RNA-seq of parent and recurrent cells grown on plastic and 0.5kpa identified PLEKHA7 significantly upregulated specifically in recurrent cells grown on 0.5 kPa substrate. PLEKHA7 was also found to be high in recurrent GBM patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provide an in vitro model system that captures the observed in vivo and clinical behavior of recurrent GBM by mimicking mechanical microenvironment and identifies PLEKHA7 as a novel potential target for recurrent GBM.

Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation.

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.

Plakophilin3 loss leads to an increase in autophagy and radio-resistance.

Loss of the desmosomal plaque protein plakophilin3 (PKP3) leads to increased tumor progression and metastasis. As metastatic tumors are often resistant to therapy, we wished to determine whether PKP3 loss led to increased radioresistance. PKP3 knockdown cells showed increased resistance to radiation in vitro and in vivo. The increase in resistance was accompanied by an increased ability to clear reactive oxygen species (ROS) and increased autophagy. The increase in autophagy was required for radioresistance and ROS clearance as inhibiting autophagy using either chloroquine or knocking down ATG3 re-sensitized the PKP3 knockdown clones to radiotherapy. These experiments suggest that autophagy inhibitors could target therapy-resistant PKP3 deficient tumors.

Loco-regional radiosensitizing nanoparticles-in-gel augments head and neck cancer chemoradiotherapy.

The therapeutic gain in loco-regionally advanced unresectable head and neck squamous cell carcinoma (HNSCC) is limited with the traditional use of concurrent chemoradiotherapy (CRT) owing to dose-limiting toxicities of systemic clinical radiosensitizers. Delivery through regional platforms is challenging due to limited drug permeation but allows spatio-temporal control of combinatorial regimens locally to overcome drug resistance. We address these challenges by developing biodegradable gellan- and lipid-based dual nanocarriers-in-ion-triggered porous mucoadhesive hydrogels for enhanced site-specific delivery of clinically relevant radiosensitizers i.e. cisplatin and paclitaxel. Interestingly, the nanoparticle-in-gel prolonged the tumor bioaccumulation of both the chemotherapeutic drugs with reduced systemic absorption, thereby improving in vivo efficacy which was confirmed by PET-CT imaging and safety as compared to systemic commercial formulations approved for HNSCC chemoradiotherapy. The nanoparticles facilitated intracellular radiosensitizer uptake and cell arrest to synergistically enhance radiation-induced DNA nicks and apoptosis. Our findings suggest the clinical potential of the present platform in the loco-regional management of HNSCC requiring curative CRT.

In Vivo Tumorigenicity Assays Using Subcutaneous and Orthotopic Xenografts in Immunodeficient Mice.

Evaluation of tumorigenic potential of medulloblastoma cell lines in vivo has been the obvious and major next step following cell line driven research for last many years. Effect of changes in expression of gene/s or efficacy of anticancer drugs on tumor initiation and/or growth can be easily assessed by injecting genetically modified cell lines in vivo or by treating in vivo xenografts of established cell lines with newer inhibitors or anticancer drugs. These studies are easy to perform and to reproduce in comparison to patient derived xenografts owing to ease in propagating, maintaining, and modifying genetic makeup of cell lines. Here we describe standardized protocols of obtaining either subcutaneous or orthotopic xenografts of medulloblastoma cell lines in immunodeficient mice. Once established, tumor growth of xenografts can be assessed during the course of experiment by either employing a simple method using Vernier caliper or technically demanding but sensitive method like in vivo bioluminescence imaging. In addition, xenograft tumors of euthanized animals can be preserved as formalin-fixed tissue specimens for further histopathological, immunohistochemical, or molecular analysis.

IGF1R-α6 integrin-S100A4 network governs the organ-specific metastasis of chemoresistant epithelial ovarian cancer cells.

Recurrent metastatic epithelial ovarian cancer (EOC) is challenging and associated with treatment limitations, as the mechanisms governing the metastatic behavior of chemoresistant EOC cells remain elusive. Using orthotopic xenograft mouse models of sensitive and acquired platinum-taxol-resistant A2780 EOC cells, we studied the mechanistic role of insulin like growth factor 1 receptor (IGF1R) signaling in the regulation of organ-specific metastasis of EOC cells undergoing acquirement of chemoresistance. Biochemical assays and organ-specific fibroblast-EOC cell co-culture were used to study the differential metastatic characteristics of sensitive vs. chemoresistant EOC cells, and the key molecule/s underlying the organ-specific homing of chemoresistant EOC cells were identified through subtractive LC/MS profiling of the co-culture secretome. The role of the identified molecule was validated through genetic/pharmacologic perturbation experiments. Acquired chemoresistance augmented organ-specific metastasis of EOC cells and enhanced lung homing, particularly for the late-stage chemoresistant cells, which was abrogated after IGF1R silencing. Escalation of chemoresistance (intrinsic and acquired) conferred EOC cells with higher adhesion toward primary lung fibroblasts, largely governed by the α6 integrin-IGF1R dual signaling axes. Subtractive analysis of the co-culture secretome revealed that interaction with lung fibroblasts induced the secretion of S100A4 from highly resistant EOC cells, which reciprocally activated lung fibroblasts. Genetic and pharmacologic inhibition of S100A4 significantly lowered distant metastases and completely abrogated lung-tropic nature of late-stage chemoresistant EOC cells. These results indicate that chemoresistance exacerbates organ-specific metastasis of EOC cells via the IGF1R-α6 integrin-S100A4 molecular network, of which S100A4 may serve as a potential target for the treatment of recurrent metastatic EOC.

Plakophilin3 loss leads to increased adenoma formation and rectal prolapse in APCmin mice.

Plakophilin3 (PKP3) loss leads to tumor progression and metastasis of colon cancer cells. The goal of this report was to determine if PKP3 loss led to increased disease progression in mice. We generated a colonocyte-specific knockout of PKP3 in APCmin mice, which led to increased adenoma formation, the formation of rectal prolapse, and a significant decrease in survival. The observed increase in rectal prolapse formation and decrease in survival correlated with an increase in the expression of Lipocalin2 (LCN2). Increased disease progression was observed even upon treatment with 5-fluorouracil (5FU). These results suggest that an increase in LCN2 expression might lead to therapy resistance and that LCN2 might serve as a potential therapeutic target in colorectal cancer.

DUSP6 regulates radiosensitivity in glioblastoma by modulating the recruitment of phosphorylated DNAPKcs at DNA double-strand breaks.

Glioblastoma (GBM) has poor median survival due to its resistance to chemoradiotherapy, which results in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is upregulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies compared to expression levels in primary GBM biopsies. Importantly, although it has been reported to be a cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse populations of GBM cell lines generated from an in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radiosensitized primary and, importantly, relapse GBM cells by inhibiting the recruitment of phosphorylated DNAPKcs (also known as PRKDC), subsequently downregulating the recruitment of phosphorylated histone H2AX (γH2AX) and 53BP1 (also known as TP53BP1). This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

Ultrasound augments on-demand breast tumor radiosensitization and apoptosis through a tri-responsive combinatorial delivery theranostic platform.

Advanced inoperable triple-negative breast cancer (TNBC) comprises aggressive tumors with a modest pathological response to neoadjuvant chemotherapy. The concomitant use of chemoradiotherapy improves the pathological response rates. However, the dose-dependent systemic toxicity of clinical radiosensitizers with poor circulation half-life and limited passive bioavailability limits their clinical utility. We address these challenges by rationally designing a stealth and tumor microenvironment responsive nano-conjugate platform for the ultrasound-mediated on-demand spatio-temporal delivery of plant flavonoid curcumin as a combinatorial regimen with clinically approved paclitaxel for the neoadjuvant chemoradiotherapy of locally advanced triple-negative breast cancer (TNBC). Interestingly, the focused application of ultrasound at the orthotopic TNBC xenograft of NOD-SCID mice facilitated the immediate infiltration of nano-conjugates at the tumor interstitium, and conferred in vivo safety over marketed paclitaxel formulation. In addition, curcumin significantly potentiated the in vivo chemoradiotherapeutic efficacy of paclitaxel upon loading into nano-conjugates. This gets further enhanced by the concurrent pulse of ultrasound, as confirmed by PET-CT imaging, along with a significant improvement in the mice survival. The quadrapeutic apoptotic effect by the combination of paclitaxel, curcumin, radiation, and ultrasound, along with a reduction in the tumor microvessel density and cell proliferation marker, confers the broad chemo-radiotherapeutic potential of this regimen for radio-responsive solid tumors, as well as metastatic niches.

Lipocalin 2 expression promotes tumor progression and therapy resistance by inhibiting ferroptosis in colorectal cancer.

Lipocalin 2 is a siderophore-binding protein that regulates iron homeostasis. Lipocalin 2 expression is elevated in multiple tumor types; however, the mechanisms that drive tumor progression upon Lipocalin 2 expression remain unclear. When Lipocalin 2 is over-expressed, it leads to resistance to 5-fluorouracil in colon cancer cell lines in vitro and in vivo by inhibiting ferroptosis. Lipocalin 2 inhibits ferroptosis by decreasing intracellular iron levels and stimulating the expression of glutathione peroxidase4 and a component of the cysteine glutamate antiporter, xCT. The increase in xCT levels is dependent on increased levels of ETS1 in Lipocalin 2 over-expressing cells. Inhibiting Lipocalin 2 function with a monoclonal antibody leads to a decrease in chemo-resistance and transformation in vitro, and a decrease in tumor progression and chemo-resistance in xenograft mouse models. Lipocalin 2 and xCT levels exhibit a positive correlation in human tumor samples suggesting that the pathway we have identified in cell lines is operative in human tumor samples. These results indicate that Lipocalin 2 is a potential therapeutic target and that the monoclonal antibody described in our study can serve as the basis for a potential therapeutic in patients who do not respond to chemotherapy.

Generation of a tissue-specific transgenic model for K8 phosphomutants: A tool to investigate the role of K8 phosphorylation during skin carcinogenesis in vivo.

Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas (SCCs), where its expression is often correlated with increased invasion, neoplastic progression, and poor prognosis. The majority of keratin 8/18 structural and regulatory functions are governed by posttranslational modifications, particularly phosphorylation. Apart from filament reorganization, cellular processes including cell cycle, cell growth, cellular stress, and apoptosis are known to be orchestrated by K8 phosphorylation at specific residues in the head and tail domains. Even though deregulation of K8 phosphorylation at two significant sites (Serine73 /Serine431 ) has been implicated in neoplastic progression of SCCs by various in vitro studies, including ours, it is reported to be highly context-dependent. Therefore, to delineate the precise role of Kereatin 8 phosphorylation in cancer initiation and progression, we have developed the tissue-specific transgenic mouse model expressing Keratin 8 wild type and phosphodead mutants under Keratin 14 promoter. Subjecting these mice to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-mediated skin carcinogenesis revealed that Keratin 8 phosphorylation may lead to an early onset of tumors compared to Keratin 8 wild-type expressing mice. Conclusively, the transgenic mouse model developed in the present study ascertained a positive impact of Keratin 8 phosphorylation on the neoplastic transformation of skin-squamous cells.

Nuclear localization of p65 reverses therapy-induced senescence.

Senescence is the arrest of cell proliferation and is a tumor suppressor phenomenon. In a previous study, we have shown that therapy-induced senescence of glioblastoma multiforme (GBM) cells can prevent relapse of GBM tumors. Here, we demonstrate that ciprofloxacin-induced senescence in glioma-derived cell lines and primary glioma cultures is defined by SA-ß-gal positivity, a senescence-associated secretory phenotype (SASP), a giant cell (GC) phenotype, increased levels of reactive oxygen species (ROS), γ-H2AX and a senescence-associated gene expression signature, and has three stages of senescence -initiation, pseudo-senescence and permanent senescence. Ciprofloxacin withdrawal during initiation and pseudo-senescence reinitiated proliferation in vitro and tumor formation in vivo Importantly, prolonged treatment with ciprofloxacin induced permanent senescence that failed to reverse following ciprofloxacin withdrawal. RNA-seq revealed downregulation of the p65 (RELA) transcription network, as well as incremental expression of SMAD pathway genes from initiation to permanent senescence. Ciprofloxacin withdrawal during initiation and pseudo-senescence, but not permanent senescence, increased the nuclear localization of p65 and escape from ciprofloxacin-induced senescence. By contrast, permanently senescent cells showed loss of nuclear p65 and increased apoptosis. Pharmacological inhibition or genetic knockdown of p65 upheld senescence in vitro and inhibited tumor formation in vivo Our study demonstrates that levels of nuclear p65 define the window of reversibility of therapy-induced senescence and that permanent senescence can be induced in GBM cells when the use of senotherapeutics is coupled with p65 inhibitors.

Sustained inhibition of PARP-1 activity delays glioblastoma recurrence by enhancing radiation-induced senescence.

Glioblastoma (GBM) is the most common primary brain tumor and is highly aggressive with a median survival of 15 months. We have previously shown that residual cells of GBM form multinucleated giant cells (MNGCs) showing a senescent phenotype, but eventually escape from therapy induced senescence (TIS), resulting in GBM recurrence. Here we demonstrate the role of PARP-1 in TIS and its recovery. We show that genetic and pharmacological inhibition of PARP-1 has an anti-proliferative effect on GBM cell lines and primary cultures derived from patient samples. Furthermore, the PARP-1 inhibitor olaparib, in combination with radiation increased MNGCs formation and senescence as assessed by ß-galactosidase activity, and macroH2A1 levels in residual cells. Additionally, we found that reduced PARP-1 activity and not protein levels in residual cells was crucial for MNGCs formation and their maintenance in the senescent state. PARP-1 activity was restored to higher levels in recurrent cells that escaped from TIS. Importantly, olaparib + radiation treatment significantly delayed recurrence in vitro as well in vivo in orthotopic GBM mouse models with a significant increase in overall survival of mice. Overall, this study demonstrates that sustained inhibition of PARP-1 activity during radiation treatment significantly delays GBM recurrence.

Inhibition of SETMAR-H3K36me2-NHEJ repair axis in residual disease cells prevents glioblastoma recurrence.

BACKGROUND: Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell-specific survival mechanism essential for GBM relapse. METHODS: Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model. RESULTS: RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival. CONCLUSIONS: We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

Histone deacetylase inhibitor pre-treatment enhances the efficacy of DNA-interacting chemotherapeutic drugs in gastric cancer.

BACKGROUND: The prognosis of gastric cancer continues to remain poor, and epigenetic drugs like histone deacetylase inhibitors (HDACi) have been envisaged as potential therapeutic agents. Nevertheless, clinical trials are facing issues with toxicity and efficacy against solid tumors, which may be partly due to the lack of patient stratification for effective treatments. AIM: To study the need of patient stratification before HDACi treatment, and the efficacy of pre-treatment of HDACi as a chemotherapeutic drug sensitizer. METHODS: The expression activity of class 1 HDACs and histone acetylation was examined in human gastric cancer cells and tissues. The potential combinatorial regime of HDACi and chemotherapy drugs was defined on the basis of observed drug binding assays, chromatin remodeling and cell death. RESULTS: In the present study, the data suggest that the differential increase in HDAC activity and the expression of class 1 HDACs are associated with hypo-acetylation of histone proteins in tumors compared to normal adjacent mucosa tissue samples of gastric cancer. The data highlights for the first time that pre-treatment of HDACi results in an increased amount of DNA-bound drugs associated with enhanced histone acetylation, chromatin relaxation and cell cycle arrest. Fraction-affected plots and combination index-based analysis show that pre-HDACi chemo drug combinatorial regimes, including valproic acid with cisplatin or oxaliplatin and trichostatin A with epirubicin, exhibit synergism with maximum cytotoxic potential due to higher cell death at low combined doses in gastric cancer cell lines. CONCLUSION: Expression or activity of class 1 HDACs among gastric cancer patients present an effective approach for patient stratification. Furthermore, HDACi therapy in pre-treatment regimes is more effective with chemotherapy drugs, and may aid in predicting individual patient prognosis.