María-Dolores Chiara

Overexpression of Focal Adhesion Kinase in Head and Neck Squamous Cell Carcinoma Is Independent of fak Gene Copy Number

The development of human malignancies can involve the aberrant regulation of intracellular signal transduction pathways that regulate cell-extracellular matrix interactions. Purpose: In the current study, we aimed to evaluate focal adhesion kinase (FAK) at both genetic and protein expression levels in head and neck squamous cell carcinomas (HNSCC) and to explore the prognostic significance of FAK. Experimental Design: A total of 211 tissue specimens, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, were analyzed using immunohistochemistry. The fak gene dosage was determined in 33 tumors. Correlations among DNA, protein, and clinicopathologic variables were analyzed. Results: FAK protein was overexpressed in HNSCCs compared with corresponding normal mucosa. High expression levels were found in 62% of the samples. Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions. All lymph node metastases examined showed FAK overexpression, with significant correlation with the expression in matched primary tumor. DNA copy number ratios for fak were higher in 39% of the tumors compared with normal mucosa. However, elevated FAK expression did not correlate with gains on DNA level, and not all cases with an amplification of the fak gene displayed protein overexpression. Similar data were obtained in five HNSCC-derived cell lines, in which FAK mRNA levels were precisely correlated with FAK protein levels. FAK protein overexpression in tumors correlated with nodal metastases. Conclusions: These findings suggest an involvement of FAK in the onset and progression of HNSCC and provide an insight into a mechanism of FAK activation alternative to gene amplification.

Identification of a Signaling Axis HIF-1α/MicroRNA-210/ISCU Independent of SDH Mutation That Defines a Subgroup of Head and Neck Paragangliomas

BACKGROUNDnHead and neck paragangliomas (HNPGLs) are rare tumors associated with the parasympathetic nervous system. Most are sporadic, but about one third result from germline mutations in succinate dehydrogenase (SDH) genes (SDHB, SDHC, SDHD, SDHA, or SDHAF2). Although a molecular connection between SDH dysfunction and tumor development is still unclear, the most accepted hypothesis proposes a central role of the pseudohypoxic pathway. SDH dysfunction induces abnormal stabilization of the hypoxia-inducible factors (HIFs) that regulate target genes involved in proliferation, apoptosis, angiogenesis, and metabolism. The involvement of these pathways in the development of sporadic HNPGLs is presently unknown.nnnOBJECTIVEnTo get some insights into the hypoxic/pseudohypoxic molecular basis of HNPGLs, we attempted to define the gene, microRNA (miRNA), and HIF-1α expression patterns that distinguish tumors from normal paraganglia tissue.nnnDESIGNnGenome microarray and TaqMan low-density arrays were used to analyze gene and miRNA expression, respectively, in 17 HNPGL tumor tissues and three normal human carotid bodies. Twelve HNPGLs were used for validation of data. HIF-1α, SDHB, and iron-sulfur cluster scaffold protein (ISCU) protein expression was analyzed by immunohistochemistry.nnnRESULTSnWe found activation of a canonical HIF-1α-related gene expression signaling only in a subset of HNPGLs from patients that did not harbor germline or somatic SDH mutations. The pseudohypoxic signature consisted in the overexpression of both HIF-1α-target genes and the HIF-1α-inducible miRNA, miR-210, and down-regulation of the miR-210 target gene, ISCU1/2. A decreased level of the iron-sulfur-containing protein SDHB was found by immunohistochemical analysis performed in two of these tumors.nnnCONCLUSIONSnCollectively, this study unveiled a putative signaling axis of HIF-1α/miRNA-210/ISCU in a subset of HNPGLs that could have an impact on SDHB protein stability by a mechanism independent of SDH mutations, thus providing a foundation to better understand the functional interplay between HIF, miR-210, and mitochondria and its relevance in the pathogenesis of HNPGLs.

Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas

BackgroundCytogenetic and gene expression analyses in head and neck squamous cell carcinomas (HNSCC) have allowed identification of genomic aberrations that may contribute to cancer pathophysiology. Nevertheless, the molecular consequences of numerous genetic alterations still remain unclear.MethodsTo identify novel genes implicated in HNSCC pathogenesis, we analyzed the genomic alterations present in five HNSCC-derived cell lines by array CGH, and compared high level focal gene amplifications with gene expression levels to identify genes whose expression is directly impacted by these genetic events. Next, we knocked down TRPC6, one of the most highly amplified and over-expressed genes, to characterize the biological roles of TRPC6 in carcinogenesis. Finally, real time PCR was performed to determine TRPC6 gene dosage and mRNA levels in normal mucosa and human HNSCC tissues.ResultsThe data showed that the HNSCC-derived cell lines carry most of the recurrent genomic abnormalities previously described in primary tumors. High-level genomic amplifications were found at four chromosomal sites (11q21-q22.2, 18p11.31-p11.21, 19p13.2-p13.13, and 21q11) with associated gene expression changes in selective candidate genes suggesting that they may play an important role in the malignant behavior of HNSCC. One of the most dramatic alterations of gene transcription involved the TRPC6 gene (located at 11q21-q22.2) which has been recently implicated in tumour invasiveness. siRNA-induced knockdown of TRPC6 expression in HNSCC-derived cells dramatically inhibited HNSCC-cell invasion but did not significantly alter cell proliferation. Importantly, amplification and concomitant overexpression of TRPC6 was also found in HNSCC tumour samples.ConclusionsAltogether, these data show that TRPC6 is likely to be a target for 11q21–22.2 amplification that confers enhanced invasive behavior to HNSCC cells. Therefore, TRPC6 may be a promising therapeutic target in the treatment of HNSCC.

Modelo murino ortotópico de carcinoma epidermoide de cabeza y cuello

Resumen Objetivo Desarrollar un modelo animal capaz de reproducir de un modo fidedigno el comportamiento de los carcinomas epidermoides de cabeza y cuello (CECC). Este modelo es necesario para la comprension de los mecanismos moleculares responsables de la genesis y progresion de estos tumores, asi como para la evaluacion de nuevos tratamientos. Material y metodos Veinte ratones atimicos nu/nu se sometieron a la inoculacion intraoral y submucosa de una linea celular derivada de un carcinoma epidermoide glotico. Resultados Dieciocho ratones (90%) desarrollaron carcinomas epidermoides localmente agresivos, con invasion del musculo y del tejido conjuntivo adyacentes. En todos ellos se encontro crecimiento perineural. Cuatro tumores (22%) presentaron destruccion osea y el 22% invasion vascular. Todos los animales que desarrollaron tumores presentaron invasion de los vasos linfaticos y metastasis ganglionares regionales. Ningun animal desarrollo metastasis hematogenas. Conclusiones Presentamos un modelo murino de CECC con capacidad para generar metastasis y que reproduce en multiples aspectos el comportamiento de estos tumores en humanos.

Increase in gene dosage is a mechanism of HIF-1α constitutive expression in head and neck squamous cell carcinomas

The HIF‐1α protein plays a key role in the cellular response to hypoxia via transcriptional regulation of genes involved in erythropoiesis, angiogenesis, and metabolism. Overexpression of HIF‐1α is commonly found in solid tumors in significant association with increased patient mortality and resistance to therapy. The predominant mode of HIF‐1α regulation by hypoxia occurs at the level of protein stability. In addition to hypoxia, HIF‐1α protein stability and synthesis is regulated by nonhypoxic signals such as inactivation of tumor suppressors and activation of oncogenes. Here, we show that an increase in gene dosage may contribute to HIF‐1α mRNA and protein overexpression in a nonhypoxic environment in head and neck squamous cell carcinomas (HNSCC). Increased HIF‐1α gene dosage was found in one out of five HNSCC‐derived cell lines and three out of 27 HNSCC primary tumors. Significantly, increased gene dosage in those samples was associated with high HIF‐1α mRNA and protein levels. Normoxic overexpression of HIF‐1α protein in HNSCC‐derived cell lines was also paralleled by higher expression levels of HIF‐1α target genes. Array CGH analysis confirmed the copy number increase of HIF‐1α gene and revealed that the gene is contained within a region of amplification at 14q23‐q24.2 both in the cell line and primary tumors. In addition, FISH analysis revealed the presence of 11–13 copies on a tetraploid background in SCC2 cells. These data suggest that increased HIF‐1α gene dosage is a mechanism of HIF‐1α protein overexpression in HNSCC that possibly prepares the cells for a higher activity in an intratumoral hypoxic environment.

Identification of Somatic VHL Gene Mutations in Sporadic Head and Neck Paragangliomas in Association With Activation of the HIF-1α/miR-210 Signaling Pathway

CONTEXTnHead and neck paragangliomas (HNPGLs) arise from parasympathetic paraganglias and 35% to 45% are hereditary caused by mutations in succinate dehydrogenase (SDH) genes. The connection between SDH and tumor development is unclear. The most accepted hypothesis proposes a central role for the pseudohypoxic (pHx) pathway activated by hypoxia-inducible factor (HIF). Paradoxically, we showed that activation of HIF in HNPGLs is restricted to a subset of HNPGLs lacking SDH mutations. These tumors overexpress HIF-1α protein and target genes and the HIF-inducible microRNA miR-210 (pHx-HNPGLs).nnnOBJECTIVEnThe present study aimed at unraveling the SDH-independent mechanisms involved in the activation of HIF in HNPGLs.nnnDESIGNnThe VHL gene was analyzed in 53 tumors by gene sequencing, multiplex-ligation-dependent probe amplification, and quantitative PCR. The miR-210, HIF-1α, and CA9 levels were used as markers of the pHx gene signature. Meta-analysis of the transcriptome of pHx-HNPGLs was performed using the Oncomine platform. Assays in cells lacking functional pVHL and HIF-1α were performed to analyze the role of pVHL/HIF-1α on miR-210 expression.nnnRESULTSnWe identified, for the first time, somatic VHL mutations in HNPGLs. These were found in 2 of 4 pHx-HNPGLs with concomitant loss of heterozygosity in one of them; but not in non-pHx-HNPGLs. Meta-analysis of the transcriptome of pHx-HNPGLs revealed that these tumors are highly related to clear cell renal cell carcinoma. Cell-based assays showed that loss of pVHL lead to upregulation of miR-210 mainly via HIF-1α activation.nnnCONCLUSIONSnVHL, involved in tumorigenesis of PGLs and clear cell renal cell carcinomas, may be an important player in the pathogenesis of sporadic HNPGLs via activation of an HIF-1α/miR-210 pHx pathway.

Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes

Tunneling nanotubes (TnTs) are thin channels that temporally connect nearby cells allowing the cell-to-cell trafficking of biomolecules and organelles. The presence or absence of TnTs in human neoplasms and the mechanisms of TnT assembly remains largely unexplored. In this study, we have identified TnTs in tumor cells derived from squamous cell carcinomas (SCC) cultured under bi-dimensional and tri-dimensional conditions and also in human SCC tissues. Our study demonstrates that TnTs are not specific of epithelial or mesenchymal phenotypes and allow the trafficking of endosomal/lysosomal vesicles, mitochondria, and autophagosomes between both types of cells. We have identified focal adhesion kinase (FAK) as a key molecule required for TnT assembly via a mechanism involving the MMP-2 metalloprotease. We have also found that the FAK inhibitor PF-562271, which is currently in clinical development for cancer treatment, impairs TnT formation. Finally, FAK-deficient cells transfer lysosomes/autophagosomes to FAK-proficient cells via TnTs which may represent a novel mechanism to adapt to the stress elicited by impaired FAK signaling. Collectively, our results strongly suggest a link between FAK, MMP-2, and TnT, and unveil new vulnerabilities that can be exploited to efficiently eradicate cancer cells.

Clinically relevant HIF-1α-dependent metabolic reprogramming in oropharyngeal squamous cell carcinomas includes coordinated activation of CAIX and the miR-210/ISCU signaling axis, but not MCT1 and MCT4 upregulation

Metabolic reprogramming is a very heterogeneous phenomenon in cancer. It mostly consists on increased glycolysis, lactic acid formation and extracellular acidification. These events have been associated to increased activity of the hypoxia inducible factor, HIF-1α. This study aimed at defining the metabolic program activated by HIF-1α in oropharyngeal squamous cell carcinomas (SCC) and assessing its clinical impact. Global gene/miRNA expression was analyzed in SCC-derived cells exposed to hypoxia. Expression of HIF-1α, the carbonic anhydrase CAIX, and the lactate/H+ transporters MCT1 and MCT4 were analyzed by immunohistochemistry in 246 SCCs. Cell-based analysis revealed that HIF-1α-driven metabolic program includes over-expression of glycolytic enzymes and the microRNA miR-210 coupled to down-regulation of its target, the iron-sulfur cluster assembly protein, ISCU. pH-regulator program entailed over-expression of CAIX, but not MCT1 or MCT4. Accordingly, significant overlapping exists between over-expression of HIF-1α and CAIX, but not HIF-1α and MCT1 or MCT4, in tumor cells. Increased miR-210 and concomitant decreased ISCU RNA levels were found in ~40% of tumors and this was significantly associated with HIF-1α and CAIX, but not MCT1 or MCT4, over-expression. HIF-1α and/or CAIX over-expression was associated with high recurrence rate and low overall survival of surgically treated patients. By contrast, clinically significant correlations were not found in tumors with MCT1 or MCT4 over-expression. This is the first study that provides in vivo evidences of coordinated activation of HIF-1α, CAIX, miR-210 and ISCU in carcinoma and association with poor prognosis, a finding with important implications for the development of metabolic-targeting therapies against hypoxia.

In vitro study of normoxic epidermal growth factor receptor–induced hypoxia-inducible factor-1-alpha, vascular endothelial growth factor, and BNIP3 expression in head and neck squamous cell carcinoma cell lines: Implications for anti–epidermal growth factor receptor therapy

We previously showed that activation of epidermal growth factor receptor (EGFR) induces hypoxia inducible factor‐1α (HIF‐1α) in head and neck squamous cell carcinoma (HNSCC) cells. In this study, we have furthered this by investigating the mechanism of HIF‐1α activation by epidermal growth factor (EGF) and its association with the sensitivity to gefitinib.

Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate.

The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.