Susceptibility Of Cancer Cells Research Articles

Regression of apoptosis-resistant colorectal tumors by induction of necroptosis in mice.

Cancer cells often acquire capabilities to evade cell death induced by current chemotherapeutic treatment approaches. Caspase-8, a central initiator of death receptor-mediated apoptosis, for example, is frequently inactivated in human cancers via multiple mechanisms such as mutation. Here, we show an approach to overcome cell death resistance in caspase-8-deficient colorectal cancer (CRC) by induction of necroptosis. In both a hereditary and a xenograft mouse model of caspase-8-deficient CRC, second mitochondria-derived activator of caspase (SMAC) mimetic treatment induced massive cell death and led to regression of tumors. We further demonstrate that receptor-interacting protein kinase 3 (RIP3), which is highly expressed in mouse models of CRC and in a subset of human CRC cell lines, is the deciding factor of cancer cell susceptibility to SMAC mimetic-induced necroptosis. Thus, our data implicate that it may be worthwhile to selectively evaluate the efficacy of SMAC mimetic treatment in CRC patients with caspase-8 deficiency in clinical trials for the development of more effective personalized therapy.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression.

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy.

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Tumor immunotherapy: drug-induced neoantigens (xenogenization) and immune checkpoint inhibitors.

More than 40 years ago, we discovered that novel transplantation antigens can be induced in vivo or in vitro by treating murine leukemia with dacarbazine. Years later, this phenomenon that we called “Chemical Xenogenization” (CX) and more recently, “Drug-Induced Xenogenization” (DIX), was reproduced by Thierry Boon with a mutagenic/carcinogenic compound (i.e. N-methyl-N’-nitro-N-nitrosoguanidine). In both cases, the molecular bases of DIX rely on mutagenesis induced by methyl adducts to oxygen-6 of DNA guanine. In the present review we illustrate the main DIX-related immune-pharmacodynamic properties of triazene compounds of clinical use (i.e. dacarbazine and temozolomide).In recent years, tumor immunotherapy has come back to the stage with the discovery of immune checkpoint inhibitors (ICpI) that show an extraordinary immune-enhancing activity. Here we illustrate the salient biochemical features of some of the most interesting ICpI and the up-to-day status of their clinical use. Moreover, we illustrate the literature showing the direct relationship between somatic mutation burden and susceptibility of cancer cells to host’s immune responses.When DIX was discovered, we were not able to satisfactorily exploit the possible presence of triazene-induced neoantigens in malignant cells since no device was available to adequately enhance host’s immune responses in clinical settings. Today, ICpI show unprecedented efficacy in terms of survival times, especially when elevated mutation load is associated with cancer cells. Therefore, in the future, mutation-dependent neoantigens obtained by appropriate pharmacological intervention appear to disclose a novel approach for enhancing the therapeutic efficacy of ICpI in cancer patients.

Critical role of HMGA proteins in cancer cell chemoresistance.

The high-mobility group A (HMGA) proteins are frequently overexpressed in human malignancies and correlate with the presence of metastases and reduced patient survival. Here, we highlight the main studies evidencing a critical role of HMGA in chemoresistance, mainly by activating Akt signaling, impairing p53 activity, and regulating the expression of microRNAs that target genes involved in the susceptibility of cancer cells to antineoplastic agents. Therefore, these studies account for the association of HMGA overexpression with patient poor outcome, indicating the impairment of HMGA as a fascinating perspective for effectively improving cancer therapy.

Modulation of Cytotoxicity by Transcription-Coupled Nucleotide Excision Repair Is Independent of the Requirement for Bioactivation of Acylfulvene.

Bioactivation as well as DNA repair affects the susceptibility of cancer cells to the action of DNA-alkylating chemotherapeutic drugs. However, information is limited with regard to the relative contributions of these processes to the biological outcome of metabolically activated DNA alkylating agents. We evaluated the influence of cellular bioactivation capacity and DNA repair on cytotoxicity of the DNA alkylating agent acylfulvene (AF). We compared the cytotoxicity and RNA synthesis inhibition by AF and its synthetic activated analogue iso-M0 in a panel of fibroblast cell lines with deficiencies in transcription-coupled (TC-NER) or global genome nucleotide excision repair (GG-NER). We related these data to the inherent bioactivation capacity of each cell type on the basis of mRNA levels. We demonstrated that specific inactivation of TC-NER by siRNA had the largest positive impact on AF activity in a cancer cell line. These findings establish that transcription-coupled DNA repair reduces cellular sensitivity to AF, independent of the requirement for bioactivation.

Astragalin-induced cell death is caspase-dependent and enhances the susceptibility of lung cancer cells to tumor necrosis factor by inhibiting the NF-кB pathway.

Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. Here, we demonstrate that the flavonoid astragalin (AG), also known as kaempferol-3-O-β-D-glucoside, induces cell death. This was prevented by the caspase inhibitors z-DEVD-FMK and z-LEHD-FMK. AG-induced cell death was associated with an increase in the Bax:Bcl-2 ratio and amplified by the inhibition of extracellular signal-regulated kinase (ERK)-1/2 and Akt signaling. Meanwhile, AG suppressed LPS-induced NF-κB activation. Additional studies revealed that AG inhibited tumor necrosis factor-alpha (TNFα)-induced NF-κB activity. AG also potentiated TNFα-induced apoptosis in A549 cells. Furthermore, using a mouse xenograft model, we demonstrated that AG suppressed tumor growth and induced cancer cell apoptosis in vivo. Taken together, these results suggest that AG may be a promising cancer therapeutic drug that warrants further investigation into its potential clinical applications.

Dual Functional Mesoporous Silicon Nanoparticles Enhance the Radiosensitivity of VPA in Glioblastoma

Radiotherapy is a critical strategy and standard adjuvant approach to glioblastoma treatment. One of the major challenges facing radiotherapy is to minimize radiation damage to normal tissue without compromising therapeutic effects on cancer cells. Various agents and numerous approaches have been developed to improve the therapeutic index of radiotherapy. Among them, radiosensitizers have attracted much attention because they selectively increase susceptibility of cancer cells to radiation and thus enhance biological effectiveness of radiotherapy. However, clinical translation of radiosensitizers has been severely limited by their potential toxicity to normal tissue. Recent advances in nanomedicine offer an opportunity to overcome this hindrance. In this study, a dual functional mesoporous silica nanoparticle (MSN) formulation of the valproic acid (VPA) radiosensitizer was developed, which specifically recognized folic acid–overexpressing cancer cells and released VPA conditionally in acidic turmeric microenvironment. The efficacy of this targeted and pH-responsive VPA nanocarrier was evaluated as compared to VPA treatment approach in two cell lines: rat glioma cells C6 and human glioma U87. Compared to VPA treatment, targeted VPA-MSNs not only potentiated the toxic effects of radiation and led to a higher rate of cell death but also enhanced inhibition on clonogenic assay. More interestingly, these effects were further accentuated by VPA-MSNs at low pH values. Western blot analysis showed that the effects were mediated via enhanced apoptosis-inducing effects. Our results suggest that the adjunctive use of VPA-MSNs may enhance the effectiveness of radiotherapy in glioma treatment by lowering the radiation doses required to kill cancer cells and thereby minimize collateral damage to healthy adjacent tissue.

The influence of cell and nanoparticle properties on heating and cell death in a radiofrequency field.

The ability of nanoparticles to either direct heating or increase susceptibility of cancer cells to radiofrequency (RF) energy remains controversial, as is the impact of cell attributes on susceptibility of cells to RF-induced cell death. This manuscript examines the impact of nanoparticle charge, size, and cellular localization on RF-induced cell death and the influence of nanoparticles on the heating rates of water and biologically-relevant media. Susceptibility of immortalized or primary cells to RF energy and the impact of cell size are also examined. The ability to selectively modulate RF heating rates in specific biological locations or in specific cell populations would enhance the therapeutic potential of RF therapy.

An invitro study on the effects of the combination of salinomycin with cisplatin on human gastric cancer cells.

The present study aimed to investigate the anticancer effects of cisplatin (DDP) combined with salinomycin (SAL) on the gastric cancer cell line SGC-7901, as well as to explore the mechanisms underlying their actions. An MTT assay was used to evaluate the inhibitory effects of SAL, DDP and their combination on gastric cancer cell proliferation. Morphological alterations of cancer cells following treatment were observed under an inverted phase-contrast microscope and a fluorescence microscope. Cell cycle progression and apoptosis were analyzed using flow cytometry. The expression of nuclear factor (NF)-κB p65 and Fas protein ligand (L) in cancer cells was assessed using immunocytochemistry. The present results demonstrated that the combination of SAL and DDP significantly inhibited the proliferation (P<0.05) and altered the morphological characteristics of SGC-7901 cells, thus suggesting that SAL may enhance the susceptibility of gastric cancer cells to DDP. In addition, treatment with a combination of SAL and DDP resulted in S phase-arrest and increased the apoptotic rate of SGC-7901 cells. Furthermore, marked FasL upregulation and NF-κB p65 downregulation were observed in cancer cells treated with the combination of SAL and DDP. The results of the present study demonstrated that the combination of SAL and DDP induced the apoptosis of human gastric cancer cells, and suggested that the underlying mechanism may involve the upregulation of FasL and downregulation of NF-κB p65.

Shutting down the power supply for DNA repair in cancer cells.

Phosphoglycerate mutase 1 (PGAM1) functions in glycolysis. In this issue, Qu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607008) show that PGAM1 inactivation leads to nucleotide depletion, which causes defective homologous recombination-mediated DNA repair, suggesting that targeting metabolic enzymes increases cancer cell susceptibility to DNA damaging agents.

Peroxiporin Expression Is an Important Factor for Cancer Cell Susceptibility to Therapeutic H2O2: Implications for Pharmacological Ascorbate Therapy

Cancer cell toxicity to therapeutic H2O2 varies widely depending on cell type. Interestingly, it has been observed that different cancer cell types have varying peroxiporin expression. We hypothesize that variation in peroxiporin expression can alter cell susceptibility to therapeutic H2O2 concentrations. Here, we silence peroxiporin aquaporin-3 (AQP3) on the pancreatic cancer cell line MIA PaCa-2 and compare clonogenic survival response to the wild-type. The results showed a significantly higher surviving fraction in the clonogenic response for siAQP3 MIA PaCa-2 cells at therapeutic H2O2 doses (P < 0.05). These results suggest that peroxiporin expression is significant in modulating the susceptibility of cancer cells to ascorbate therapy.

S4S8-RPA phosphorylation as an indicator of cancer progression in oral squamous cell carcinomas.

Oral cancers are easily accessible compared to many other cancers. Nevertheless, oral cancer is often diagnosed late, resulting in a poor prognosis. Most oral cancers are squamous cell carcinomas that predominantly develop from cell hyperplasias and dysplasias. DNA damage is induced in these tissues directly or indirectly in response to oncogene-induced deregulation of cellular proliferation. Consequently, a DNA Damage response (DDR) and a cell cycle checkpoint is activated. As dysplasia transitions to cancer, proteins involved in DNA damage and checkpoint signaling are mutated or silenced decreasing cell death while increasing genomic instability and allowing continued tumor progression. Hyperphosphorylation of Replication Protein A (RPA), including phosphorylation of Ser4 and Ser8 of RPA2, is a well-known indicator of DNA damage and checkpoint activation. In this study, we utilize S4S8-RPA phosphorylation as a marker for cancer development and progression in oral squamous cell carcinomas (OSCC). S4S8-RPA phosphorylation was observed to be low in normal cells, high in dysplasias, moderate in early grade tumors, and low in late stage tumors, essentially supporting the model of the DDR as an early barrier to tumorigenesis in certain types of cancers. In contrast, overall RPA expression was not correlative to DDR activation or tumor progression. Utilizing S4S8-RPA phosphorylation to indicate competent DDR activation in the future may have clinical significance in OSCC treatment decisions, by predicting the susceptibility of cancer cells to first-line platinum-based therapies for locally advanced, metastatic and recurrent OSCC.

The TLR Adaptor Protein MyD88 Mediates Cell Sensitivity to HDAC Inhibitors through a Cytokine-Dependent Mechanism

IntroductionHistone deacetylase inhibitors (HDI) are promising new agents for the treatment of haematological malignancy including lymphoma. HDI have potent anti-proliferative effects in cell-based studies; in the clinical setting, however, the picture is quite variable, and the outcome of HDI therapy is complex and difficult to predict.The toll-like receptor (TLR) adaptor protein MyD88 (Myeloid Differentiation Primary Response Gene 88) is expressed at high levels in haematopoietic tissues and has recently been found to be the target for somatic mutation in lymphoid malignancies. In particular, substitution of the amino acid leucine to proline at position 265 (MyD88 L265P mutation) is estimated to be prevalent in over 90% of cases of Waldenstrom's macroglobulinaemia and around 37% of diffuse large B cell lymphoma (DLBCL) of the ABC subtype.Methods and ResultsUsing an unbiased genome-wide loss-of-function screen (Fotheringham et al, 2009), MyD88 was identified as a key regulator and candidate biomarker which mediates the susceptibility of cancer cells to HDI: We show here in mechanism of action studies that expression of MyD88 can influence sensitivity of various cancer cell lines to treatment with an HDI. Short-term siRNA silencing of MyD88 reduced HDI sensitivity of U2OS osteosarcoma cells (as assessed by immunoblotting and FACS/propidium iodide staining), whereas over-expression of MyD88 and MyD88 L265P led to enhanced sensitivity.Given its role in TLR-signalling, we measured NFκB activity and pro-inflammatory cytokine expression, which were shown to be increased (in particular IL6 levels) when MyD88 was stably or transiently overexpressed in U2OS cells; co-expression of mutated MyD88 L265P augmented this effect. In DLBCL lymphoma cell lines of the ABC subtype, those cells expressing endogenous wild-type MyD88 (RIVA) were found to be less sensitive to HDI treatment compared to those cells carrying a heterozygous MyD88 L265P mutation (HBL-1). MyD88-mutated cells again showed higher levels of IL6 expression on cytokine analysis. Transient knockdown of endogenous MyD88 in ABC DLBCL cells resulted in decreased HDI sensitivity, as did disruption of the TLR/MyD88/NFκB signalling pathway by blocking either the TLR receptor, MyD88 aggregation or IL6 secretion. Immunohistochemical analysis of tumour tissue microarrays from patients with DLBCL carried out in conjunction with this work showed higher expression of MyD88 compared to patients with prostate or colorectal carcinoma. Finally, using a mass spectroscopy approach, we were able to show that MyD88 is acetylated, and that it can be directly deacetylated by the cytoplasmic histone deacetylase HDAC6.Discussion and translational significanceCollectively our findings show that in cultured cells, high levels of MyD88 expression are associated with increased sensitivity to HDAC inhibition, whereas low level expression coincides with decreased sensitivity. We hypothesize that MyD88, in its role as adaptor protein in TLR signalling, plays an important role in regulating the expression and autocrine action of pro-inflammatory cytokines such as IL6 which contribute to the sensitization of cancer cells to HDI. The central role played by MyD88 in this mechanism highlights the importance of TLR signalling and innate immunity, and more generally the inflammatory properties of the extra-cellular tumour microenvironment in mediating the effects of HDAC inhibitors on malignant cells. Therefore, both expression level of MyD88 and MyD88 mutation status could serve as a potential biomarker for HDI sensitivity in patient selection. DisclosuresNo relevant conflicts of interest to declare.

PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Erratum to: Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance. The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells. There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy. Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies.

Abstract 5175: Mutational and copy number profiling of cancer-related genes in 26 human tumor xenografts and their correlations with antitumor drug sensitivities

Abstract Background: Tumor responses to antitumor drugs are variable, but predicting these responses is important when selecting effective chemotherapy treatments. Our aim was to identify variations or alterations in gene copy number that influence cancer cells’ susceptibilities to standard chemotherapeutic agents. Methods: Twenty-six human cancer cell lines representing the five main tumor types were subcutaneously implanted into nude mice and tested for sensitivity to fluorinated pyrimidines (UFT, TS-1, 5’-DFUR, and capecitabine), CDDP, CPT-11, and paclitaxel. The cell lines included lung (AOI, LC-11, Lu-99, LX-1, LC-6, Lu-134, Lu-130), colon (KM12C, KM12C/FU, HCT-15, COL-1, CO-3), pancreas (PAN-3, PAN-4, PAN-12, H-48, MIAPaCa-2, BxPC-3), gastric (SC-2, ST-40, 4-1ST, SC-4) and breast (MC-2, MX-1, MDA-MB-435SHM, MDA-MD-231). Genomic DNA was prepared from frozen tumor tissues. Mutations in 48 genes from the TruSeq Amplicon Cancer Panel were screened using the MiSeq system (Illumina, San Diego, CA). Somatic copy number alterations were analyzed by high-density SNP arrays (Affymetrix, Santa Clara, CA). Results: Of the 225 amplicons (187 non-overlapping regions) in the cancer panel, 86% achieved a minimum average sequencing depth of 1000X and the average coverage across all target regions was 5374X. In 26 tumors, sequencing detected 55 somatic mutations in 18 out of 48 cancer related genes of high prognostic or therapeutic significance, such as TP53, APC, PTEN, and SMAD4. Mutation frequencies across 26 xenografts were 73.1% (TP53), 38.5% (KRAS), 15.4% (APC), 11.5% (SMAD4 and RET), 7.7% (BRAF, GNAS, and PTEN), and 3.8% (CTNNB1, GNAQ, HNF1A, HRAS, IDH1, KIT, NOTCH1, PIK3CA, SMO, and STK11). Tumor xenografts with TP53 mutations were significantly less sensitive to CDDP and CPT-11 than wild-type cell lines (P&amp;lt;0.05). The APC mutation conferred resistance to paclitaxel. Copy number gain was observed at 23.1% (KRAS), 15.1% (EGFR), and 11.5% (JAK2 and CDK2NA). Copy number loss was observed at 30.8% (CDK2NA), 19.2% (SMAD4), and 15.4% (PTEN). Cell lines with more copies of CDK2NA and JAK2 were more sensitive to CDDP, while cell lines with fewer copies of PTEN were more sensitive to CDDP. Similarly, copy number gain of CDH1 conferred resistance to UFT, while copy number gain of KRAS sensitized tumors to 5’-DFUR. The copy number of 48 genes determined by the GeneChip array moderately agreed with those estimated by local GC-content adjusted coverage profiles in the sequencing analysis (average Pearson's correlation coefficient = 0.66, 95%CI = 0.54-0.69). Conclusions: Integrated analysis of mutational profiling and gene copy number may be useful to elucidate candidate genes influencing susceptibility of cancer cells to antitumor drugs. Citation Format: Takashi Kobunai, Kenta Tsunekuni, Kazuaki Matsuoka, Hiroshi Tsukihara, Teiji Takechi. Mutational and copy number profiling of cancer-related genes in 26 human tumor xenografts and their correlations with antitumor drug sensitivities. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5175.

Abstract 2114: Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells

Abstract ST6Gal-I is a sialyltransferase that adds α2-6 linked sialic acids to cell surface proteins as they pass through the trans-Golgi. Sialic acids, being negatively charged, are able to alter the function of selected cell surface receptors, which leads to dysregulation of various downstream cellular pathways. Overexpression of ST6Gal-I has been observed in various cancers including ovarian and pancreatic cancer. We have previously shown that knockdown of ST6Gal-I expression increases ovarian cancer cell susceptibility to the chemotherapeutic drug cisplatin. In the present study we further investigate whether resistance to additional drugs such as gemcitabine, the front line treatment for pancreatic cancer, is affected by ST6Gal-I activity. MiaPaCa-2 and BxPC3 pancreatic cancer cell lines, having high ST6Gal-I expression, were employed for our studies. To elucidate the mechanistic role of ST6Gal-I in gemcitabine resistance we created stable ST6Gal-I knockdown lines of MiaPaCa-2 and BxPC3 cells. Gemcitabine induced cell death was more pronounced in the knockdown cell lines, indicated by heightened activation of caspase-3. Gemcitabine is metabolized to a nucleoside analogue, gemcitabine triphosphate, which induces apoptosis by promoting DNA damage. Increased single stand DNA damage in the knockdown cells was confirmed using alkaline comet assay. Gemcitabine treatment led to greater activation of DNA damage markers and response elements (γH2AX, phospho CHK1, phospho CHK2), along with increased levels of cleaved caspase-3 in the knockdown cells as compared to empty vector control cells. We next developed a stable gemcitabine resistant MiaPaCa-2 cell line by growing parental cells in gemcitabine. We selected for the population of cells that survived and were able to replicate in gemcitabine containing media. ST6Gal-I levels were found to be increased in the stable gemcitabine resistant lines relative to parental cell lines, suggesting that cells with high ST6Gal-I expression selectively survive gemcitabine treatment. By measuring the levels of genes involved in activation over those responsible for inactivation of gemcitabine, we were able to obtain a gemcitabine sensitivity predictive ratio for MiaPaCa-2 and BxPC3 ST6Gal-I knockdown, empty vector, parental and stable gemcitabine resistant cell lines. This ratio has been described previously in literature as a metric for gauging gemcitabine resistance. According to the ratios obtained, ST6Gal-I knockdown enhanced drug sensitivity whereas high expression of ST6Gal-I offered protection against gemcitabine induced apoptosis. Collectively these data indicate that upregulation of ST6Gal-I imparts tumor cell survival through prevention of gemcitabine induced DNA damage. Citation Format: Asmi Chakraborty, Matthew Schultz, Hoa Q. Trummell, James A. Bonner, Susan Bellis. Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2114.

Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer.

BackgroundColorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance.MethodsThe expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells.ResultsThere were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy.ConclusionIncreased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0948-z) contains supplementary material, which is available to authorized users.

Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection.

Interferons (IFNs) induce anti-viral programs, regulate immune responses, and exert anti-proliferative effects. To escape anti-tumorigenic effects of IFNs, malignant cells attenuate JAK/STAT signaling and expression of IFN stimulated genes (ISGs). Such attenuation may enhance the susceptibility of tumor cells to oncolytic virotherapy. Here we studied genetic and epigenetic mechanisms of interference with JAK/STAT signaling and their contribution to susceptibility of prostate cancer cells to viral infection. Bioinformatics analysis of gene-expression in cohorts of prostate cancer patients revealed genetic and epigenetic interference with the IFN program. To correlate lack of IFN signaling and susceptibility to viral infection and oncolysis; we employed LNCaP prostate cancer cells as cellular model, and the human metapneumovirus and the epizootic hemorrhagic disease virus as infectious agents. In LNCaP cells, JAK1 is silenced by bi-allelic inactivating mutations and epigenetic silencing, which also silences ISGs. Chemical inhibition of epigenetic silencing partially restored IFN-sensitivity, induced low levels of expression of selected ISGs and attenuated, but failed to block, viral infection and oncolysis. Since viral infection was not blocked by epigenetic modifiers, and these compounds may independently-induce anti-tumor effects, we propose that epigenetic modifiers and virotherapy are compatible in treatment of prostate tumors defective in JAK1 expression and IFN signaling.