Gemcitabine-induced epithelial-mesenchymal transition-like changes sustain chemoresistance of pancreatic cancer cells of mesenchymal-like phenotype.

Growing body of evidence suggests that epithelial-mesenchymal transition (EMT) is a critical process in tumor progression and chemoresistance in pancreatic cancer (PC). The aim of this study was to analyze the role of EMT-like changes in acquisition of resistance to gemcitabine in pancreatic cells of the mesenchymal or epithelial phenotype. Therefore, chemoresistant BxPC-3, Capan-2, Panc-1, and MiaPaca-2 cells were selected by chronic exposure to increasing concentrations of gemcitabine. We show that gemcitabine-resistant Panc-1 and MiaPaca-2 cells of mesenchymal-like phenotype undergo further EMT-like molecular changes mediated by ERK-ZEB-1 pathway, and that inhibition of ERK1/2 phosphorylation or ZEB-1 expression resulted in a decrease in chemoresistance. Conversely, gemcitabine-resistant BxPC-3 and Capan-2 cells of epithelial-like phenotype did not show such typical EMT-like molecular changes although the expression of the tight junction marker occludin could be found decreased. In pancreatic cancer patients, high ZEB-1 expression was associated with tumor invasion and tumor budding. In addition, tumor budding was essentially observed in patients treated with neoadjuvant chemotherapy. These findings support the notion that gemcitabine treatment induces EMT-like changes that sustain invasion and chemoresistance in PC cells.

γ-Tubulin⁻γ-Tubulin Interactions as the Basis for the Formation of a Meshwork.

In cytoplasm, protein γ-tubulin joins with various γ-tubulin complex proteins (GCPs) to form a heterotetramer γ-tubulin small complex (γ-TuSC) that can grow into a ring-shaped structure called the γ-tubulin ring complex (γ-TuRC). Both γ-TuSC and γ-TuRC are required for microtubule nucleation. Recent knowledge on γ-tubulin with regard to its cellular functions beyond participation in its creation of microtubules suggests that this protein forms a cellular meshwork. The present review summarizes the recognized functions of γ-tubulin and aims to unite the current views on this protein.

Hubbing the Cancer Cell.

Oncogenic transformation drives adaptive changes in a growing tumor that affect the cellular organization of cancerous cells, resulting in the loss of specialized cellular functions in the polarized compartmentalization of cells. The resulting altered metabolic and morphological patterns are used clinically as diagnostic markers. This review recapitulates the known functions of actin, microtubules and the γ-tubulin meshwork in orchestrating cell metabolism and functional cellular asymmetry.

The γ-tubulin meshwork assists in the recruitment of PCNA to chromatin in mammalian cells.

Changes in the location of γ-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of γ-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the γ-tubulin meshwork assists in the recruitment of PCNA to chromatin. Also, decreased levels of γ-tubulin reduce the nuclear pool of PCNA. In addition, the γ-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a γ-tubulin-PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and γ-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the γ-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.

Non-Canonical Functions of the Gamma-Tubulin Meshwork in the Regulation of the Nuclear Architecture.

The nuclear architecture describes the organization of the various compartments in the nucleus of eukaryotic cells, where a plethora of processes such as nucleocytoplasmic transport, gene expression, and assembly of ribosomal subunits occur in a dynamic manner. During the different phases of the cell cycle, in post-mitotic cells and after oncogenic transformation, rearrangements of the nuclear architecture take place, and, among other things, these alterations result in reorganization of the chromatin and changes in gene expression. A member of the tubulin family, γtubulin, was first identified as part of a multiprotein complex that allows nucleation of microtubules. However, more than a decade ago, γtubulin was also characterized as a nuclear protein that modulates several crucial processes that affect the architecture of the nucleus. This review presents the latest knowledge regarding changes that arise in the nuclear architecture of healthy cells and under pathological conditions and, more specifically, considers the particular involvement of γtubulin in the modulation of the biology of the nuclear compartment.

Toward the Discovery of a Novel Class of YAP⁻TEAD Interaction Inhibitors by Virtual Screening Approach Targeting YAP⁻TEAD Protein⁻Protein Interface.

Intrinsically disordered protein YAP (yes-associated protein) interacts with TEADs transcriptional factors family (transcriptional enhancer associated domain) creating three interfaces. Interface 3, between the Ω-loop of YAP and a shallow pocket of TEAD was identified as the most important TEAD zone for YAP-TEAD interaction. Using the first X-ray structure of the hYAP50⁻71-hTEAD1209⁻426 complex (PDB 3KYS) published in 2010, a protein-protein interaction inhibitors-enriched library (175,000 chemical compounds) was screened against this hydrophobic pocket of TEAD. Four different chemical families have been identified and evaluated using biophysical techniques (thermal shift assay, microscale thermophoresis) and in cellulo assays (luciferase activity in transfected HEK293 cells, RTqPCR in MDA-MB231 cells). A first promising hit with micromolar inhibition in the luciferase gene reporter assay was discovered. This hit also decreased mRNA levels of TEAD target genes.

Molecular Features of the YAP Inhibitor Verteporfin: Synthesis of Hexasubstituted Dipyrrins as Potential Inhibitors of YAP/TAZ, the Downstream Effectors of the Hippo Pathway.

Porphyrin derivatives, in particular verteporfin (VP), a photosensitizer initially designed for cancer therapy, have been identified as inhibitors of the YAP-TEAD interaction and transcriptional activity. Herein we report the efficient convergent synthesis of the dipyrrin half of protoporphyrin IX dimethyl ester (PPIX-DME), in which the sensitive vinyl group was created at the final stage by a dehydroiodination reaction. Two other dipyrrin derivatives were synthesized, including dipyrrin 19 [(Z)-2-((3,5-dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-3,5-dimethyl-4-vinyl-1H-pyrrole], containing two vinyl groups. We found that VP and dipyrrin 19 showed significant inhibitory effects on TEAD transcriptional activity in MDA-MB-231 human breast cancer cells, whereas other compounds did not show significant changes. In addition, we observed a marked decrease in both YAP and TAZ levels following VP treatment, whereas dipyrrin 19 treatment primarily decreased the levels of YAP and receptor kinase AXL, a downstream target of YAP. Together, our data suggest that, due to their chemical structures, porphyrin- and dipyrrin-related derivatives can directly target YAP and/or TAZ proteins and inhibit TEAD transcriptional activity.

Non-Photoinduced Biological Properties of Verteporfin.

BACKGROUND: Verteporfin is a porphyrinic photosensitizer clinically used for the photodynamic treatment of age-related macular degeneration. It has been identified almost simultaneously as a YAP/TEAD and an autophagosome inhibitor. Over the last few years, YAP (TAZ), the downstream effectors of the Hippo pathway, have emerged as promising anticancer targets, as shown by several experimental lines of evidence, showing the overproduction of YAP in several cancers. However, YAP was also found to be closely connected to autophagy, mitochondria and reactive oxygen/nitrogen species. We herein, review the recent studies where VP was used without photoactivation as a YAP/TEAD inhibitor or protein oligomerization promoter, focusing on its effects on the YAP/TEAD gene targets and other biomarkers related to autophagy. RESULTS: Since the identification of VP as YAP/TEAD inhibitor, several in vitro and in vivo studies have revealed the new potential of this molecule in different cancers, where YAP is overexpressed. However, detailed structural information about its interaction with YAP is still lacking. Concomitantly, VP was identified as autophagosome inhibitor by promoting oligomerization of p62. Moreover, VP proves to be tumor-selective proteotoxic (by oligomerization of p62, STAT3) in colorectal cancer. Knowledge on the biological properties of the only YAP inhibitor available to date is vital for its pharmacological use on cellular and animal models. CONCLUSION: VP is a multi-target drug interacting with several proteins implicated in major cellular processes. Although this does not impact its clinical use, VP does not seem to be the ideal drug for pharmacological inhibitions of YAP/TEAD.

Regulation of cellular quiescence by YAP/TAZ and Cyclin E1 in colon cancer cells: Implication in chemoresistance and cancer relapse.

Our aim was to decipher the role and clinical relevance of the YAP/TAZ transcriptional coactivators in the regulation of the proliferation/quiescence balance in human colon cancer cells (CCC) and survival after 5FU-based chemotherapy. The prognostic value of YAP/TAZ on tumor relapse and overall survival was assessed in a five-year follow-up study using specimens of liver metastases (n = 70) from colon cancer patients. In 5FU-chemoresistant HT29-5F31 and -chemosensitive HCT116 and RKO CCC, a reversible G0 quiescent state mediated by Cyclin E1 down-regulation was induced by 5FU in 5F31 cells and recapitulated in CCC by either YAP/TAZ or Cyclin E1 siRNAs or the YAP inhibitor Verteporfin. Conversely, the constitutive active YAPdc-S127A mutant restricted cellular quiescence in 5FU-treated 5F31 cells and sustained high Cyclin E1 levels through CREB Ser-133 phosphorylation and activation. In colon cancer patients, high YAP/TAZ level in residual liver metastases correlated with the proliferation marker Ki-67 (p < 0.0001), high level of the YAP target CTGF (p = 0.01), shorter disease-free and overall survival (p = 0.008 and 0.04, respectively). By multivariate analysis and Cox regression model, the YAP/TAZ level was an independent factor of overall (Hazard ratio [CI 95%] 2.06 (1.02-4.16) p = 0.045) and disease-free survival (Hazard ratio [CI 95%] 1.98 (1.01-3.86) p = 0.045). Thus, YAP/ TAZ pathways contribute to the proliferation/quiescence switch during 5FU treatment according to the concerted regulation of Cyclin E1 and CREB. These findings provide a rationale for therapeutic interventions targeting these transcriptional regulators in patients with residual chemoresistant liver metastases expressing high YAP/TAZ levels.

Colon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis.

PURPOSE: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. RESULTS: We show that a clonal 5F31 cell population, resistant to 1 µmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). CONCLUSIONS: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.