Study of adaptive responses to drug treatments in pancreatic cancer cells

One of the most important and unsolved problems in the therapy of pancreatic tumors is the development of resistance to pharmacological treatments. Inefficacy of therapies leads to the progression of these cancers, worsening the quality of life and shortening the lifespan. Tumors that interest the pancreas are quite different from each other, depending on the cell type by which it origins and on the alterations of pancreatic functionality. They may be classified in two groups: endocrine tumors, which arise from pancreatic endocrine cells, and carcinomas, which derive from cells with exocrine functions. Cancers derived from these two groups are very different in terms of prognosis. Indeed, pancreatic endocrine tumors are defined as “indolent”, due to their slow rate of growth and to the long survival of patients in most of the cases. On the contrary, pancreatic ductal carcinoma is considered one of the most lethal tumors, due to the inefficacy of chemotherapeutic treatment and by the short survival of patient. In both cases, tumors are incurable in most patients. The pharmacological approaches used for the treatment of endocrine or ductal tumors are different: in endocrine tumors, which have a slow progression rate, inhibitors of cell growth are used in the majority of cases, while in ductal carcinomas, which have a faster progression, chemotherapy with agents that impair replication of cancerous cells is preferred. The aim of the work presented in this thesis was to investigate the mechanisms at the basis of drug resistance in pancreatic tumors. The first part of the work is focused on the study of endocrine tumors and it proposes a novel strategy to counteract the problem of resistance of pharmacological treatment with Everolimus, using in vitro models. The second part of the work concerns the investigation of the molecular features of pancreatic ductal cancer cells, and how these mechanisms may be involved in the malignity of cancer cells and in the acquisition of resistance to chemotherapy

Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first-line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing (AS) of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence-free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favor the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, upregulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to upregulation of PTBP1 and modulation of PKM AS in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy.Oncogene advance online publication, 3 August 2015; doi:10.1038/onc.2015.270

Alternative polyadenylation of ZEB1 promotes its translation during genotoxic stress in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. The standard chemotherapeutic drug, gemcitabine, does not offer significant improvements for PDAC management due to the rapid acquisition of drug resistance by patients. Recent evidence indicates that epithelial-to-mesenchymal transition (EMT) of PDAC cells is strictly associated to early metastasization and resistance to chemotherapy. However, it is not exactly clear how EMT is related to drug resistance or how chemotherapy influences EMT. Herein, we found that ZEB1 is the only EMT-related transcription factor that clearly segregates mesenchymal and epithelial PDAC cell lines. Gemcitabine treatment caused upregulation of ZEB1 protein through post-transcriptional mechanisms in mesenchymal PDAC cells within a context of global inhibition of protein synthesis. The increase in ZEB1 protein correlates with alternative polyadenylation of the transcript, leading to shortening of the 3' untranslated region (UTR) and deletion of binding sites for repressive microRNAs. Polysome profiling indicated that shorter ZEB1 transcripts are specifically retained on the polysomes of PDAC cells during genotoxic stress, while most mRNAs, including longer ZEB1 transcripts, are depleted. Thus, our findings uncover a novel layer of ZEB1 regulation through 3'-end shortening of its transcript and selective association with polysomes under genotoxic stress, strongly suggesting that PDAC cells rely on upregulation of ZEB1 protein expression to withstand hostile environments

Cerebral hemodynamics on MR perfusion images before and after bypass surgery in patients with giant intracranial aneurysms

Preoperative assessment of the anatomy and dynamics of cerebral circulation for patients with giant intracranial aneurysm can improve both outcome prediction and therapeutic approach. The aim of our study was to use perfusion MR imaging to evaluate cerebral hemodynamics in such patients before and after extraintracranial high-flow bypass surgery. METHODS: Five patients with a giant aneurysm of the intracranial internal carotid artery underwent MR studies before, 1 week after, and 1 month after high-flow bypass surgery. We performed MR and digital subtraction angiography, and conventional and functional MR sequences (diffusion and perfusion). Surgery consisted of middle cerebral artery (MCA)-internal carotid artery bypass with saphenous vein grafts (n = 4) or MCA-external carotid artery bypass (n = 1). RESULTS: In four patients, MR perfusion study showed impaired hemodynamics in the vascular territory supplied by the MCA of the aneurysm side, characterized by significantly reduced mean cerebral blood flow (CBF), whereas mean transit time (MTT) and regional cerebral blood volume (rCBV) were either preserved, reduced, or increased. After surgery, angiography showed good canalization of the bypass graft. MR perfusion data obtained after surgery showed improved cerebral hemodynamics in all cases, with a return of CBF index (CBFi), MTT, and rCBV to nearly normal values. CONCLUSION: Increased MTT with increased or preserved rCBV can be interpreted as a compensatory vasodilatory response to reduced perfusion pressure, presumably from compression and disturbed flow in the giant aneurysmal sac. When maximal vasodilation has occurred, however, the brain can no longer compensate for diminished perfusion by vasodilation, and rCBV and CBFi diminish. Bypass surgery improves hemodynamics, increasing perfusion pressure and, thus, CBFi. Perfusion MR imaging can be used to evaluate cerebral hemodynamics in patients with intracranial giant aneurysm.BACKGROUND AND PURPOSE: Preoperative assessment of the anatomy and dynamics of cerebral circulation for patients with giant intracranial aneurysm can improve both outcome prediction and therapeutic approach. The aim of our study was to use perfusion MR imaging to evaluate cerebral hemodynamics in such patients before and after extraintracranial high-flow bypass surgery. METHODS: Five patients with a giant aneurysm of the intracranial internal carotid artery underwent MR studies before, 1 week after, and 1 month after high-flow bypass surgery. We performed MR and digital subtraction angiography, and conventional and functional MR sequences (diffusion and perfusion). Surgery consisted of middle cerebral artery (MCA)-internal carotid artery bypass with saphenous vein grafts (n = 4) or MCA-external carotid artery bypass (n = 1). RESULTS: In four patients, MR perfusion study showed impaired hemodynamics in the vascular territory supplied by the MCA of the aneurysm side, characterized by significantly reduced mean cerebral blood flow (CBF), whereas mean transit time (MTT) and regional cerebral blood volume (rCBV) were either preserved, reduced, or increased. After surgery, angiography showed good canalization of the bypass graft. MR perfusion data obtained after surgery showed improved cerebral hemodynamics in all cases, with a return of CBF index (CBFi), MTT, and rCBV to nearly normal values. CONCLUSION: Increased MTT with increased or preserved rCBV can be interpreted as a compensatory vasodilatory response to reduced perfusion pressure, presumably from compression and disturbed flow in the giant aneurysmal sac. When maximal vasodilation has occurred, however, the brain can no longer compensate for diminished perfusion by vasodilation, and rCBV and CBFi diminish. Bypass surgery improves hemodynamics, increasing perfusion pressure and, thus, CBFi. Perfusion MR imaging can be used to evaluate cerebral hemodynamics in patients with intracranial giant aneurysm

HNRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPMmotifswere significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment

Molecular pathogenesis and targeted therapy of sporadic pancreatic neuroendocrine tumors

Over the past few years, knowledge regarding the molecular pathology of sporadic pancreatic neuroendocrine tumors (PNETs) has increased substantially, and a number of targeted agents have been tested in clinical trials in this tumor type. For some of these agents there is a strong biological rationale. Among them, the mammalian target of rapamycin inhibitor Everolimus and the antiangiogenic agent Sunitinib have both been approved for the treatment of PNETs. However, there is lack of knowledge regarding biomarkers able to predict their efficacy, and mechanisms of resistance. Other angiogenesis inhibitors, such as Pazopanib, inhibitors of Src, Hedgehog or of PI3K might all be useful in association or sequence with approved agents. On the other hand, the clinical significance, and potential for treatment of the most common mutations occurring in sporadic PNETs, in the MEN-1 gene and in ATRX and DAXX, remains uncertain. The present paper reviews the main molecular changes occurring in PNETs and how they might be linked with treatment options

3-Aroyl-1,4-diarylpyrroles inhibit chronic myeloid leukemia cell growth through an interaction with tubulin

We designed 3-aroyl-1,4-diarylpyrrole (ARDAP) derivatives as potential anticancer agents having different substituents at the 1- or 4-phenyl ring. ARDAP compounds exhibited potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARDAP derivative 10 inhibited the proliferation of BCR/ABL-expressing KU812 and LAMA84 cells from chronic myeloid leukemia (CML) patients in blast crisis and of hematopoietic cells ectopically expressing the imatinib mesylate (IM)-sensitive KBM5-WT or its IM-resistant KBM5-T315I mutation. Compound 10 minimally affected the proliferation of normal blood cells, indicating that it may be a promising agent to overcome broad tyrosine kinase inhibitor resistance in relapsed/refractory CML patients. Compound 10 significantly decreased CML proliferation by inducing G2/M phase arrest and apoptosis via a mitochondria-dependent pathway. ARDAP 10 augmented the cytotoxic effects of IM in human CML cells. Compound 10 represents a robust lead compound to develop tubulin inhibitors with potential as novel treatments for CML

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer

New pyrrole derivatives with potent tubulin polymerization inhibiting activity as anticancer agents including hedgehog-dependent cancer

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway