Carmine Carbone

Modulation of Pancreatic Cancer Chemoresistance by Inhibition of TAK1

BACKGROUND TGF-β-activated kinase-1 (TAK1), a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor κB (NF-κB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer. METHODS NF-κB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n = 40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. The results of in vitro proliferation were analyzed for statistical significance of differences by nonlinear regression analysis; differences in mouse survival were determined using a log-rank test. All statistical tests were two-sided. RESULTS AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-κB activity than did their respective control cell lines (relative luciferase activity: AsPc-1, mean = 0.18, 95% confidence interval [CI] = 0.10 to 0.27; control, mean = 3.06, 95% CI = 2.31 to 3.80; MDAPanc-28, mean = 0.30, 95% CI = 0.13 to 0.46; control, mean = 4.53, 95% CI = 3.43 to 5.63; both P < .001). TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC(50) between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells (P < .001). In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden (gemcitabine vs gemcitabine plus LYTAK1, P = .03) and prolonged survival duration (median survival: gemcitabine, 82 days vs gemcitabine plus LYTAK1, 122 days; hazard ratio = 0.334, 95% CI = 0.027 to 0.826, P = .029). CONCLUSIONS The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.

HDAC inhibitor vorinostat enhances the antitumor effect of gefitinib in squamous cell carcinoma of head and neck by modulating ErbB receptor expression and reverting EMT.

Potentiation of epidermal growth factor receptor (EGFR) inhibitors is required in squamous cell carcinoma of head and neck (SCCHN) to improve their therapeutic index. We demonstrated that the histone deacetylase inhibitor vorinostat in combination with the EGFR tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration, and invasion as well as induction of apoptosis in SCCHN cells, including cells resistant to gefitinib. We provided evidence suggesting that differential modulation of ErbB receptors together with reversion of epithelial‐to‐mesenchymal transition (EMT) by vorinostat represent mechanistic bases for the observed synergism. We demonstrated in epithelial CAL27 cells expressing EGFR, ErbB2, and ErbB3 that vorinostat downregulated the expression and signaling of all three receptors. In gefitinib‐resistant KB and Hep‐2 cells, both of which had undergone EMT and expressed very low levels of ErbB3, vorinostat reverted the mesenchymal phenotype by inducing both E‐cadherin and ErbB3 and downregulating vimentin as well as EGFR and ErbB2. Both transcriptional and post‐translational mechanisms were involved in the modulation of ErbB receptors by vorinostat. Attenuation of all ErbB transcripts in CAL27 cells as well as induction of ErbB3 transcript in Hep‐2 and KB cells was seen upon vorinostat treatment. We showed that vorinostat induced ubiquitination of EGFR and ErbB2 and targeted them predominantly to lysosome‐degradation in all cell lines, while the induction of ErbB3‐ubiquitination in CAL27 cells led to proteasomes‐degradation. Overall, this study suggests that the vorinostat/gefitinib combination represents a promising therapeutic strategy that warrants further clinical evaluation in SCCHN, including tumors intrinsically resistant to EGFR‐inhibitors. J. Cell. Physiol. 226: 2378–2390, 2011.

NF-κB as a target for pancreatic cancer therapy

Introduction: Pancreatic cancer is the fourth leading cause of adult cancer mortality in the USA. It represents one of the greatest challenges in cancer treatment. The NF-κB transcriptional factors are constitutively activated in the majority of pancreatic cancers and are involved in the regulation of numerous aspects of tumor development and progression. NF-κB and the signaling cascades that regulate its activity have thus become attractive targets for novel therapeutic approaches for pancreatic cancer. Areas covered: This review describes and discusses the most important advances in the comprehension of the complex molecular biology of NF-κB, as well as the development of novel NF-κB-targeting strategies for the treatment of pancreatic cancer. Expert opinion: Although the inhibition of NF-κB, especially when combined with more classic chemotherapeutic drugs, could be a promising therapeutic strategy, direct targeting NF-κB still faces important challenges. In the future, targeting nonredundant cytosolic mediators of the activation of NF-κB – such as TNF receptor associated factor family member-associated NF-κB activator -binding kinase 1 (TBK1) and TGF-beta activated kinase 1 (TAK1) – could represent a better approach to inhibit key processes in pancreatic tumor cells and make a difference for this devastating disease.

Anti-VEGF Treatment Resistant Pancreatic Cancers Secrete Proinflammatory Factors that Contribute to Malignant Progression by Inducing an EMT cell phenotype

Purpose: The resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. There are currently no validated predictive biomarkers for selecting which cancer patients will benefit from antiangiogenic therapy. Also lacking are resistance biomarkers that can identify which escape pathways should be targeted after tumors develop resistance to VEGF treatment. Recent studies showed that anti-VEGF treatment can make tumor cells more aggressive and metastatic. However, the mechanisms and mediators of this are unidentified. Therefore, we aimed this study at directly identifying the tumor cell–initiated mechanisms responsible for the resistance of pancreatic cancer to anti-VEGF treatment. Experimental Design: We established and validated two murine models of human pancreatic cancer resistant to the VEGF-specific antibody bevacizumab in vivo. We used a genome-wide analysis to directly identify which tumor-secreted factors were overexpressed by pancreatic cancer cells that were resistant to anti-VEGF treatment. Results: Rather than direct proangiogenic factors, we identified several proinflammatory factors that were expressed at higher levels in cells resistant to anti-VEGF treatment than in treatment-sensitive control cells. These proinflammatory factors acted in a paracrine manner to stimulate the recruitment of CD11b+ proangiogenic myeloid cells. Also, we found that secreted factors overexpressed by anti-VEGF treatment-resistant pancreatic cancer cells acted in an autocrine manner to induce epithelial-to-mesenchymal transition (EMT) and were thus responsible for increased aggressiveness of bevacizumab-resistant pancreatic tumors. Conclusions: Our results identified proinflammatory factors and EMT markers as potential biomarkers for selecting patients with pancreatic cancer for antiangiogenic therapy. Clin Cancer Res; 17(17); 5822–32. ©2011 AACR.

Mechanisms of resistance to chemotherapeutic and anti-angiogenic drugs as novel targets for pancreatic cancer therapy

Pancreatic cancer remains one of the most lethal and poorly understood human malignancies and will continue to be a major unsolved health problem in the 21st century. Despite efforts over the past three decades to improve diagnosis and treatment, the prognosis for patients with pancreatic cancer is extremely poor with or without treatment, and incidence rates are virtually identical to mortality rates. Although advances have been made through the identification of relevant molecular pathways in pancreatic cancer, there is still a critical, unmet need for the translation of these findings into effective therapeutic strategies that could reduce the intrinsic drug resistance of this disease and for the integration of these molecularly targeted agents into established combination chemotherapy and radiotherapy regimens in order to improve patients’ survival. Tumors are heterogeneous cellular entities whose growth and progression depend on reciprocal interactions between genetically altered neoplastic cells and a non-neoplastic microenvironment. To date, most of the mechanisms of resistance studied have been related to tumor cell-autonomous signaling pathways. However, recent data suggest a putative important role of tumor microenvironment in the development and maintenance of resistance to classic chemotherapeutic and targeted therapies. This present review is meant to describe and discuss some of the most important advances in the comprehension of the tumor cell-autonomous and tumor microenvironment-related molecular mechanisms responsible for the resistance of pancreatic cancer to the proapoptotic activity of the classic chemotherapeutic agents and to the most novel anti-angiogenic drugs. We present some of the emerging therapeutic targets for the modulation of this resistant phenotype.

Acquired resistance to zoledronic acid and the parallel acquisition of an aggressive phenotype are mediated by p38-MAP kinase activation in prostate cancer cells

The nitrogen-containing bisphosphonates (N-BP) zoledronic acid (ZOL) inhibits osteoclast-mediated bone resorption, and it is used to prevent skeletal complications from bone metastases. ZOL has also demonstrated anticancer activities in preclinical models and, recently, in cancer patients, highlighting the interest in determining eventual mechanisms of resistance against this agent. In our study, we selected and characterised a resistant subline of prostate cancer (PCa) cells to better understand the mechanisms, by which tumour cells can escape the antitumour effect of ZOL. DU145R80-resistant cells were selected in about 5 months using stepwise increasing concentrations of ZOL from DU145 parental cells. DU145R80 cells showed a resistance index value of 5.5 and cross-resistance to another N-BP, pamidronate, but not to the non-nitrogen containing BP clodronate. Notably, compared with DU145 parental cells, DU145R80 developed resistance to apoptosis and anoikis, as well as overexpressed the anti-apoptotic protein Bcl-2 and oncoprotein c-Myc. Moreover, DU145R80 cells underwent epithelial to mesenchymal transition (EMT) and showed increased expression of the metalloproteases MMP-2/9, as well as increased invading capability. Interestingly, compared with DU145, DU145R80 cells also increased the gene expression and protein secretion of VEGF and the cytokines Eotaxin-1 and IL-12. At the molecular level, DU145R80 cells showed strong activation of the p38-MAPK-dependent survival pathway compared with parental sensitive cells. Moreover, using the p38-inhibitor SB203580, we completely reversed the resistance to ZOL, as well as EMT marker expression and invasion. Furthermore, SB203580 treatment reduced the expression of VEGF, Eotaxin-1, IL-12, MMP-9, Bcl-2 and c-Myc. Thus, for the first time, we demonstrate that the p38-MAPK pathway can be activated under continuous extensive exposure to ZOL in PCa cells and that the p38-MAPK pathway has a critical role in the induction of resistance, as well as in the acquisition of a more aggressive and invasive phenotype.

Tissue transglutaminase: a new target to reverse cancer drug resistance

Cancer resistance mechanisms, which result from intrinsic genetic alterations of tumor cells or acquired genetic and epigenetic changes, limit the long-lasting benefits of anti-cancer treatments. Tissue transglutaminase (TG2) has emerged as a putative gene involved in tumor cell drug resistance and evasion of apoptosis. Although some reports have indicated that TG2 can suppress tumor growth and enhance the growth inhibitory effects of anti-tumor agents, several studies have presented both pro-survival and anti-apoptotic roles for TG2 in malignant cells. Increased TG2 expression has been found in several tumors, where it was considered a potential negative prognostic marker, and it is often associated with advanced stages of disease, metastatic spread and drug resistance. TG2 mediates drug resistance through the activation of survival pathways and the inhibition of apoptosis, but also by regulating extracellular matrix (ECM) formation, the epithelial-to-mesenchymal transition (EMT) or autophagy. Because TG2 knockdown or inhibition of TG2 enzymatic activity may reverse drug resistance and sensitize cancer cells to drug-induced apoptosis, many small molecules capable of blocking TG2 have recently been developed. Additional insight into the multifunctional nature of TG2 as well as translational studies concerning the correlation between TG2 expression, function or location and cancer behavior will aid in translating these findings into new therapeutic approaches for cancer patients.

An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients

Purpose: The majority of gastric cancer patients who achieve an initial response to trastuzumab-based regimens develop resistance within 1 year of treatment. This study was aimed at identifying the molecular mechanisms responsible for resistance. Experimental Design: A HER2+-trastuzumab sensitive NCI-N87 gastric cancer orthotopic nude mouse model was treated with trastuzumab until resistance emerged. Differentially expressed transcripts between trastuzumab-resistant and sensitive gastric cancer cell lines were annotated for functional interrelatedness by Ingenuity Pathway Analysis software. Immunohistochemical analyses were performed in pretreatment versus posttreatment biopsies from gastric cancer patients receiving trastuzumab-based treatments. All statistical tests were two-sided. Results: Four NCI-N87 trastuzumab-resistant (N87-TR) cell lines were established. Microarray analysis showed HER2 downregulation, induction of epithelial-to-mesenchymal transition, and indicated fibroblast growth factor receptor 3 (FGFR3) as one of the top upregulated genes in N87-TR cell lines. In vitro, N87-TR cell lines demonstrated a higher sensitivity than did trastuzumab-sensitive parental cells to the FGFR3 inhibitor dovitinib, which reduced expression of pAKT, ZEB1, and cell migration. Oral dovitinib significantly (P = 0.0006) reduced tumor burden and prolonged mice survival duration in N87-TR mouse models. A higher expression of FGFR3, phosphorylated AKT, and ZEB1 were observed in biopsies from patients progressing under trastuzumab-based therapies if compared with matched pretreatment biopsies. Conclusions: This study identified the FGFR3/AKT axis as an escape pathway responsible for trastuzumab resistance in gastric cancer, thus indicating the inhibition of FGFR3 as a potential strategy to modulate this resistance. Clin Cancer Res; 22(24); 6164–75. ©2016 AACR.

Rationale and clinical use of multitargeting anticancer agents

Human solid tumors contain genetically distinct subpopulations of tumor cells that can be enriched under selective pressure of specific treatments. This heterogeneous nature reflects the dynamism of drug response and it represents a fundamental driver of resistance. Moreover, the complexity of cancer disease is increased by the activity of cross-talking, redundant signaling pathways, escape pathways and compensatory events, which triggers activation of secondary growth and survival. Broad multi-targeted approaches are requested to overcome a complex, heterogeneous, and dynamic disease such as cancer.

Combined inhibition of IL1, CXCR1/2, and TGFβ signaling pathways modulates in-vivo resistance to anti-VEGF treatment.

Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin-1 (IL1), CXC receptors (CXCR)1/2 ligands, and transforming growth factor &bgr; (TGF&bgr;) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the antivascular endothelial growth factor (anti-VEGF) antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse this anti-VEGF resistance. Bevacizumab-resistant FGBR pancreatic cancer cells were treated in vitro with bevacizumab, the recombinant human IL1 receptor antagonist anakinra, the monoclonal antibody against TGF&bgr; receptor type II TR1, and a novel recombinant antibody binding CXCR1/2 ligands. The FGBR cells treated with these agents in combination had significantly higher levels of E-cadherin and lower levels of vimentin, IL6, phosphorylated p65, and SMAD2, and showed significantly lower migration rates than did their controls treated with the same agents without bevacizumab or with a single agent bevacizumab as a control. Consistently, the combination of these agents with bevacizumab reduced the FGBR tumor burden and significantly prolonged mice survival compared with bevacizumab in monotherapy. Tumors from mice receiving the combination treatment showed significantly lower expression of IL6 and phosphorylated SMAD2, higher expression of E-cadherin and lower levels of vimentin, and a significantly lower infiltration by CD11b+ cells compared with bevacizumab-treated controls. This study suggests that inhibition of IL1, CXCR1/2, and TGF&bgr; signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing epithelial–mesenchymal transition and inhibiting CD11b+ proangiogenic myeloid cells’ tumor infiltration.