Emilios Gemenetzidis

FOXM1 Upregulation Is an Early Event in Human Squamous Cell Carcinoma and it Is Enhanced by Nicotine during Malignant Transformation

Background Cancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC). Methodology/Principal Findings FOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients) consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR), expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP) array was used to ‘trace’ the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23), were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression. Conclusions/Significance This study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that aberrant upregulation of FOXM1 may be inducing genomic instability through a program of malignant transformation involving the activation of CEP55 and HELLS which may facilitate aberrant mitosis and epigenetic modifications. Our finding that FOXM1 is upregulated early during oral cancer progression renders FOXM1 an attractive diagnostic biomarker for early cancer detection and its candidate mechanistic targets, CEP55 and HELLS, as indicators of malignant conversion and progression.

Induction of Human Epithelial Stem/Progenitor Expansion by FOXM1

Stem cells are permanent residents of tissues and thought to be targets of cancer initiation. The frequent, and often early, upregulation of the FOXM1 transcription factor in the majority of human cancers suggests that it may participate in the initiation of human tumorigenesis. However, this hypothesis has not been tested. Herein, we show that targeting the ectopic expression of FOXM1 to the highly clonogenic cells of primary human keratinocytes with stem/progenitor cell properties, but not to differentiating cells, caused clonal expansion in vitro. We show, using a functional three-dimensional organotypic epithelial tissue regeneration system, that ectopic FOXM1 expression perturbed epithelial differentiation generating a hyperproliferative phenotype reminiscent of that seen in human epithelial hyperplasia. Furthermore, transcriptional expression analysis of a panel of 28 epithelial differentiation-specific genes reveals a role for FOXM1 in the suppression of epithelial differentiation. This study provides the first evidence that FOXM1 participates in an early oncogenic pathway that predisposes cells to tumorigenesis by expanding the stem/progenitor compartment and deregulating subsequent keratinocyte terminal differentiation. This finding reveals an important window of susceptibility to oncogenic signals in epithelial stem/progenitor cells prior to differentiation, and may provide a significant benefit to the design of cancer therapeutic interventions that target oncogenesis at its earliest incipient stage.

FOXM1 induces a global methylation signature that mimics the cancer epigenome in head and neck squamous cell carcinoma.

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.

Maintenance of stem cell self‐renewal in head and neck cancers requires actions of GSK3β influenced by CD44 and RHAMM

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44high but one is also ESAhigh and maintains epithelial characteristics, the other is ESAlow, has mesenchymal characteristics and is migratory. Examining CD44‐regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3β (GSK3β). We show that inhibitory phosphorylation reduces the formation of both “tumor spheres” and “holoclone” colonies, functional indicators of stemness. GSK3β inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44high/ESAhigh cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3β knockdown. These results indicate that GSK3β plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

Anti-stromal treatment together with chemotherapy targets multiple signalling pathways in pancreatic adenocarcinoma.

Stromal targeting for pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming an attractive option, due to the lack of efficacy of standard chemotherapy and increased knowledge about PDAC stroma. We postulated that the addition of stromal therapy may enhance the anti‐tumour efficacy of chemotherapy. Gemcitabine and all‐trans retinoic acid (ATRA) were combined in a clinically applicable regimen, to target cancer cells and pancreatic stellate cells (PSCs) respectively, in 3D organotypic culture models and genetically engineered mice (LSL‐KrasG12D/+;LSL‐Trp53R172H/+;Pdx‐1‐Cre: KPC mice) representing the spectrum of PDAC. In two distinct sets of organotypic models as well as KPC mice, we demonstrate a reduction in cancer cell proliferation and invasion together with enhanced cancer cell apoptosis when ATRA is combined with gemcitabine, compared to vehicle or either agent alone. Simultaneously, PSC activity (as measured by deposition of extracellular matrix proteins such as collagen and fibronectin) and PSC invasive ability were both diminished in response to combination therapy. These effects were mediated through a range of signalling cascades (Wnt, hedgehog, retinoid, and FGF) in cancer as well as stellate cells, affecting epithelial cellular functions such as epithelial–mesenchymal transition, cellular polarity, and lumen formation. At the tissue level, this resulted in enhanced tumour necrosis, increased vascularity, and diminished hypoxia. Consequently, there was an overall reduction in tumour size. The enhanced effect of stromal co‐targeting (ATRA) alongside chemotherapy (gemcitabine) appears to be mediated by dampening multiple signalling cascades in the tumour–stroma cross‐talk, rather than ablating stroma or targeting a single pathway.

Topical 4-thiothymidine is a viable photosensitiser for the photodynamic therapy of skin malignancies

Abstract The nucleoside analogue 4-thiothymidine has shown great potential in vitro as a photosensitiser for the photodynamic therapy of numerous cancer cell lines. However, the limited penetrating power of UV-A radiation, to which it responds, raises doubts as to its practical usefulness in clinical applications. We addressed this issue by studying the penetration extent of topical thiothymidine and the antiproliferative effect of its combination with UV-A radiation on ex vivo basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) skin cancer biopsies, and normal skin. Our results show that both the intralesional concentration of the drug and the intensity of UV-A radiation are sufficient to activate the molecule and cause extensive death by apoptosis of the malignant cells. Normal skin biopsies were not significantly affected by the treatment.

Invasive oral cancer stem cells display resistance to ionising radiation

There is a significant amount of evidence to suggest that human tumors are driven and maintained by a sub-population of cells, known as cancer stem cells (CSC). In the case of head and neck cancer, such cells have been characterised by high expression levels of CD44 cell surface glycoprotein, while we have previously shown the presence of two diverse oral CSC populations in vitro, with different capacities for cell migration and proliferation. Here, we examined the response of oral CSC populations to ionising radiation (IR), a front-line measure for the treatment of head and neck tumors. We show that oral CSC initially display resistance to IR-induced growth arrest as well as relative apoptotic resistance. We propose that this is a result of preferential activation of the DNA damagerepair pathway in oral CSC with increased activation of ATM and BRCA1, elevated levels of DNA repair proteins RAD52, XLF, and a significantly faster rate of DNA double-strand-breaks clearance 24 hours following IR. By visually identifying CSC sub-populations undergoing EMT, we show that EMT-CSC represent the majority of invasive cells, and are more radio-resistant than any other population in re-constructed 3D tissues. We provide evidence that IR is not sufficient to eliminate CSC in vitro, and that sensitization of CD44hi/ESAlow cells to IR, followed by secondary EMT blockade, could be critical in order to reduce primary tumor recurrence, but more importantly to be able to eradicate cells capable of invasion and distant metastasis.

GLI2 induces genomic instability in human keratinocytes by inhibiting apoptosis.

Abnormal Sonic Hedgehog signalling leads to increased transcriptional activation of its downstream effector, glioma 2 (GLI2), which is implicated in the pathogenesis of a variety of human cancers. However, the mechanisms underlying the tumorigenic role of GLI2 remain elusive. We demonstrate that overexpression of GLI2-β isoform, which lacks the N-terminal repressor domain (GLI2ΔN) in human keratinocytes is sufficient to induce numerical and structural chromosomal aberrations, including tetraploidy/aneuploidy and chromosomal translocations. This is coupled with suppression of cell cycle regulators p21WAF1/CIP1 and 14-3-3σ, and strong induction of anti-apoptotic signalling, resulting in a reduction in the ability to eliminate genomically abnormal cells. Overexpression of GLI2ΔN also rendered human keratinocytes resistant to UVB-mediated apoptosis, whereas inhibition of B-cell lymphoma 2 (BCL-2) restored endogenous (genomic instability (GIN)) and exogenous (UVB) DNA damage-induced apoptosis. Thus, we propose that ectopic expression of GLI2 profoundly affects the genomic integrity of human epithelial cells and contributes to the survival of progenies with genomic alterations by deregulating cell cycle proteins and disabling the apoptotic mechanisms responsible for their elimination. This study reveals a novel role for GLI2 in promoting GIN, a hallmark of human tumors, and identifies potential mechanisms that may provide new opportunities for the design of novel forms of cancer therapeutic strategies.

RhoC Interacts with Integrin α5β1 and Enhances Its Trafficking in Migrating Pancreatic Carcinoma Cells

Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin α5β1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin α5β1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin α5β1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin α5β1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin α5β1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.

Abstract B72: Developing drug combinations to co-target pancreatic cancer and its supporting stroma

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense desmoplastic stroma, due to the activation of pancreatic stellate cells (PSC). The activated PSC are characterized by a reduction of fat droplets, expression of α-smooth muscle actin (α-SMA), and an increased synthesis and secretion of several connective tissue components such as fibronectin (FN) and collagen type I. Aberrant stromal-epithelial interactions contribute to pancreatic cancer spread and metastasis raising the possibility that targeting the stroma may represent viable strategy for treating pancreatic cancer. Our group has shown that pancreatic cancer progression can be restrained via modulation of the stellate cells in the stroma. Methods: Organotypic cultures (OT) were constructed to mimic human PDAC. Treatment of OT was performed to mimic human chemotherapy regimen cycles with all-trans retinoic acid (ATRA), targeting PSC, and the chemotherapeutic agent Gemcitabine (GEM), targeting pancreatic cancer cells (PCC). Furthermore, KPC mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) were treated with the combination of GEM and ATRA. Appropriate controls were used for both in vivo and in vitro assays using either drug alone or vehicle alone to dissect effect of individual drug. Both in vivo and in vitro assays were studied for various surrogate endpoints for chemotherapy efficacy. Results:In the organotypic model, ATRA alone or in combination with GEM reduced cancer cell and stellate cell invasion. The same treatment also reduced cancer cell proliferation. Stellate cell activation, as measured by fibronectin production per stellate cell, was significantly reduced upon combination treatment with GEM/ATRA, in comparison to cultures treated with GEM or ATRA only. In the KPC mice model, ATRA alone or in combination with GEM caused stromal collapse. The same regimen treatment led to an increase in vascular density in the tumor area and a significant impairment of the tumor growth, when compared with mice treated with ATRA or GEM alone. The number of necrotic areas was also higher in tumors from mice treated with the combination treatment of GEM/ATRA. Further assessments are being carried out. Conclusions:The combination treatment of all-trans retinoic acid and Gemcitabine may have a synergistic detrimental effect upon pancreatic cancer progression. Citation Format: Elisabete Florencio Carapuca, Emilios Gemenetzidis, Christine Feig, David Tuveson, Hemant Kocher. Developing drug combinations to co-target pancreatic cancer and its supporting stroma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B72.