Cristina Montero-Conde

Relief of Feedback Inhibition of HER3 Transcription by RAF and MEK Inhibitors Attenuates Their Antitumor Effects in BRAF-Mutant Thyroid Carcinomas

The RAF inhibitor vemurafenib (PLX4032) increases survival in patients with BRAF-mutant metastatic melanoma, but has limited efficacy in patients with colorectal cancers. Thyroid cancer cells are also comparatively refractory to RAF inhibitors. In contrast to melanomas, inhibition of mitogen-activated protein kinase (MAPK) signaling by PLX4032 is transient in thyroid and colorectal cancer cells. The rebound in extracellular signal-regulated kinase (ERK) in thyroid cells is accompanied by increased HER3 signaling caused by induction of ERBB3 (HER3) transcription through decreased promoter occupancy by the transcriptional repressors C-terminal binding protein 1 and 2 and by autocrine secretion of neuregulin-1 (NRG1). The HER kinase inhibitor lapatinib prevents MAPK rebound and sensitizes BRAF-mutant thyroid cancer cells to RAF or MAP-ERK kinase inhibitors. This provides a rationale for combining ERK pathway antagonists with inhibitors of feedback-reactivated HER signaling in this disease. The determinants of primary resistance to MAPK inhibitors vary between cancer types, due to preferential upregulation of specific receptor tyrosine kinases, and the abundance of their respective ligands.

Tumoral and tissue-specific expression of the major human β-tubulin isotypes†

The β‐tubulins are microtubule components encoded by a multigene family, which produces slightly different proteins with complex expression patterns. Several widely used anticancer drugs base their activity on β‐tubulin binding, microtubule dynamics alteration, and cell division blockage. The expression of these drug targets in tumoral and normal cells could be of crucial importance for therapy outcome, unfortunately, the complex β‐tubulin expression patterns have been poorly characterized in human. In this study, we developed a quantitative RT‐PCR technique that accurately determines the mRNA expression of the eight human β‐tubulin isotypes, encoding class I, IIa, IIb, III, IVa, IVb, V, and VI and applied it to 21 nontumoral tissues and 79 tumor samples belonging to seven cancer types. In the nontumoral tissues, we found that, overall, TUBB (I), TUBB2C (IVb), and TUBB6 (V) were ubiquitous, TUBB1(VI) was hematopoietic cell‐specific, and TUBB2A (IIa), TUBB2B (IIb), TUBB3 (III), and TUBB4 (IVa) had high expression in brain; however, the contribution of the different isotypes to the total β‐tubulin content varied for each tissue and had a complex pattern. In tumoral tissues, most isotypes exhibited an altered expression in specific tumor types or related to tumoral characteristics. In general, TUBB3 showed a great increase in expression while TUBB6 expression was largely decreased in most tumors. Thus, normal tissues showed a complex β‐tubulin isotype distribution, which could contribute to the toxicity profile of the microtubule‐binding drugs. In addition, the specific isotypes significantly altered in tumors might represent markers for drug response.

Research Resource: Transcriptional Profiling Reveals Different Pseudohypoxic Signatures in SDHB and VHL-Related Pheochromocytomas

The six major genes involved in hereditary susceptibility for pheochromocytoma (PCC)/paraganglioma (PGL) (RET, VHL, NF1, SDHB, SDHC, and SDHD) have been recently integrated into the same neuronal apoptotic pathway where mutations in any of these genes lead to cell death. In this model, prolyl hydroxylase 3 (EglN3) abrogation plays a pivotal role, but the molecular mechanisms underlying its inactivation are currently unknown. The aim of the study was to decipher specific alterations associated with the different genetic classes of PCCs/PGLs. With this purpose, 84 genetically characterized tumors were analyzed by means of transcriptional profiling. The analysis revealed a hypoxia-inducible factor (HIF)-related signature common to succinate dehydrogenase (SDH) and von Hippel-Lindau (VHL) tumors, that differentiated them from RET and neurofibromatosis type 1 cases. Both canonical HIF-1α and HIF-2α target genes were overexpressed in the SDH/VHL cluster, suggesting that a global HIF deregulation accounts for this common profile. Nevertheless, when we compared VHL tumors with SDHB cases, which often exhibit a malignant behavior, we found that HIF-1α target genes showed a predominant activation in the VHL PCCs. Expression data from 67 HIF target genes was sufficient to cluster SDHB and VHL tumors into two different groups, demonstrating different pseudo-hypoxic signatures. In addition, VHL-mutated tumors showed an unexpected overexpression of EglN3 mRNA that did not lead to significantly different EglN3 protein levels. These findings pave the way for more specific therapeutic approaches for malignant PCCs/PGLs management based on the patients genetic alteration.

The Variant rs1867277 in FOXE1 Gene Confers Thyroid Cancer Susceptibility through the Recruitment of USF1/USF2 Transcription Factors

In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30–1.70; P = 5.9×10−9). Functional assays of rs1867277 (NM_004473.3:c.−283G>A) within the FOXE1 5′ UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/αCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

Genetics of pheochromocytoma and paraganglioma in Spanish patients.

CONTEXT The presence of familial history in pheochromocytoma/paraganglioma patients, including syndromic antecedents, leads in the majority of cases to a positive genetic testing for mutations in one of the major susceptibility genes described so far. Furthermore, it has been reported that in the absence of familial antecedents, about 11-24% of patients also carry a mutation in one of these related genes. In these cases, other clinical aspects like bilaterality, multiplicity, location of the tumors, or age at onset can help to recognize the underlying genes involved. OBJECTIVE The objective of the study was to discuss clinical criteria helpful in the genetic diagnosis, placing special emphasis on apparently sporadic cases. DESIGN Two hundred thirty-seven nonrelated probands were analyzed for the major susceptibility genes: VHL, RET, SDHB, SDHC, and SDHD. Genetic characterization included both point mutation analysis and gross deletions in the SDH genes performed by multiplex PCR. RESULTS As expected, all syndromic probands were genetically diagnosed with a mutation affecting either RET or VHL. A total of 79.1% (19 of 24) and 18.4% (31 of 168) of patients presenting with either nonsyndromic familial antecedents or apparently sporadic presentation were found to carry a mutation in one of the susceptibility genes. Finally, we found a Spanish founder effect for two mutations: SDHB c.166_170delCCTCA and SDHD c.129G>A. CONCLUSIONS Germline mutations are rare in apparently sporadic probands diagnosed after age 40 yr (3.9% in our series) and mainly involve SDHB. Therefore, we recommend prioritizing SDHB genetic testing in patients developing isolated tumors at any age, especially those with extraadrenal location or malignant behavior.

Identification of kinase fusion oncogenes in post-Chernobyl radiation-induced thyroid cancers

Exposure to ionizing radiation during childhood markedly increases the risk of developing papillary thyroid cancer. We examined tissues from 26 Ukrainian patients with thyroid cancer who were younger than 10 years of age and living in contaminated areas during the time of the Chernobyl nuclear reactor accident. We identified nonoverlapping somatic driver mutations in all 26 cases through candidate gene assays and next-generation RNA sequencing. We found that 22 tumors harbored fusion oncogenes that arose primarily through intrachromosomal rearrangements. Altogether, 23 of the oncogenic drivers identified in this cohort aberrantly activate MAPK signaling, including the 2 somatic rearrangements resulting in fusion of transcription factor ETS variant 6 (ETV6) with neurotrophic tyrosine kinase receptor, type 3 (NTRK3) and fusion of acylglycerol kinase (AGK) with BRAF. Two other tumors harbored distinct fusions leading to overexpression of the nuclear receptor PPARγ. Fusion oncogenes were less prevalent in tumors from a cohort of children with pediatric thyroid cancers that had not been exposed to radiation but were from the same geographical regions. Radiation-induced thyroid cancers provide a paradigm of tumorigenesis driven by fusion oncogenes that activate MAPK signaling or, less frequently, a PPARγ-driven transcriptional program.

Polymorphisms in cytochromes P450 2C8 and 3A5 are associated with paclitaxel neurotoxicity

Neurotoxicity is one of the most relevant dose-limiting toxicities of the anticancer drug paclitaxel. It exhibits substantial interindividual variability of unknown molecular basis, and represents one of the major challenges for the improvement of paclitaxel therapy. The extensive variability in paclitaxel clearance and metabolism lead us to investigate the association between polymorphisms in paclitaxel elimination pathway and neurotoxicity. We selected 13 relevant polymorphisms in genes encoding paclitaxel metabolizing enzymes (CYP2C8, CYP3A4 and CYP3A5) and transporters (organic anion transporting polypeptide (OATP) 1B1, OATP1B3 and P-glycoprotein) and genotyped them in 118 Spanish cancer patients treated with paclitaxel. After adjusting for age and treatment schedule, CYP2C8 Haplotype C and CYP3A5*3 were associated with protection (hazard ratio (HR) (per allele)=0.55; 95% confidence interval (CI)=0.34–0.89; P=0.014 and HR (per allele)=0.51; 95%CI=0.30–0.86; and P=0.012, respectively) and CYP2C8*3 with increased risk (HR (per allele)=1.72; 95%CI=1.05–2.82; and P=0.032). In each case, the allele causing increased paclitaxel metabolism was associated with increased neurotoxicity, suggesting an important role for metabolism and hydroxylated paclitaxel metabolites. We estimated the HR per paclitaxel-metabolism increasing allele carried across the three polymorphisms to be HR=1.64 (95% CI=1.26–2.14; P=0.0003). The results for P-glycoprotein were inconclusive, and no associations were observed for the other genes studied. The incorporation of this genetic data in treatment selection could help to reduce neurotoxicity events, thereby individualizing paclitaxel pharmacotherapy. These results warrant validation in independent series.

Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information.

Undifferentiated and poorly differentiated thyroid tumors are responsible for more than half of thyroid cancer patient deaths in spite of their low incidence. Conventional treatments do not obtain substantial benefits, and the lack of alternative approaches limits patient survival. Additionally, the absence of prognostic markers for well-differentiated tumors complicates patient-specific treatments and favors the progression of recurrent forms. In order to recognize the molecular basis involved in tumor dedifferentiation and identify potential markers for thyroid cancer prognosis prediction, we analysed the expression profile of 44 thyroid primary tumors with different degrees of dedifferentiation and aggressiveness using cDNA microarrays. Transcriptome comparison of dedifferentiated and well-differentiated thyroid tumors identified 1031 genes with >2-fold difference in absolute values and false discovery rate of <0.15. According to known molecular interaction and reaction networks, the products of these genes were mainly clustered in the MAPkinase signaling pathway, the TGF-β signaling pathway, focal adhesion and cell motility, activation of actin polymerization and cell cycle. An exhaustive search in several databases allowed us to identify various members of the matrix metalloproteinase, melanoma antigen A and collagen gene families within the upregulated gene set. We also identified a prognosis classifier comprising just 30 transcripts with an overall accuracy of 95%. These findings may clarify the molecular mechanisms involved in thyroid tumor dedifferentiation and provide a potential prognosis predictor as well as targets for new therapies.

Overexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis

Therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) are limited due to lack of effective treatments. Thus, there is a need to thoroughly characterize the pathways of molecular pathogenesis and to identify potential targets for therapy in MTC. Since epidermal growth factor receptor (EGFR) seems to play a crucial role for RET activation, a key feature of MTCs, and several promising EGFR/vascular endothelial growth factor receptor 2 (VEGFR2)-targeted drugs have been developed, the present study was designed to investigate whether these proteins are altered in MTCs. We used a well-characterized series of 153 MTCs to evaluate EGFR activation by sequencing and FISH analysis, and to perform EGFR and VEGFR2 immunohistochemistry. EGFR tyrosine kinase domain mutations were not a feature of MTCs; however, EGFR polysomy and a strong EGFR expression were detected in 15 and 13% of the tumors respectively. Interestingly, EGFR was significantly overexpressed in metastases compared with primary tumors (35 vs 9%, P=0.002). We also studied whether specific RET mutations were associated with EGFR status, and found a decrease in EGFR polysomies (P=0.006) and a tendency towards lower EGFR expression for the most aggressive RET mutations (918, 883). Concerning VEGFR2, metastasis showed a higher expression than primary tumors (P=2.8 x 10(-8)). In this first study investigating the relationship between EGFR, RET, and VEGFR2 in a large MTC series, we found an activation of EGFR and VEGFR2 in metastasis, using both independent and matched primary/metastasis samples. This suggests that some MTC patients may benefit from existing anti-EGFR/VEFGR2 therapies, although additional preclinical and clinical evidence is needed.

Gross SDHB deletions in patients with paraganglioma detected by multiplex PCR: A possible hot spot?

Pheochromocytoma and paraganglioma are rare neuroendocrine tumors that arise in the adrenal medulla and the extra‐adrenal paraganglia, respectively. Inheritance of these tumors is mainly a result of mutations affecting the VHL, RET, NF1, and SDH genes. Germ‐line mutations of the SDH genes have been found to account for nearly 10% of apparently sporadic cases. Nevertheless, alterations other than point mutations have not yet been well characterized. In this study, we investigated the frequency of gross SDH deletions in 24 patients who tested negative for point mutations and had at least one of the recommended features for genetic testing. For this purpose, we used a technique that is easy to implement in the lab to specifically detect gross deletions affecting SDHB, SDHC, and SDHD. We identified 3 heterozygous SDHB deletions (3/24) in 3 independent cases with paraganglioma: 1 whole SDHB deletion and 2 deletions exclusively affecting exon 1. These latter mutations match the unique gross deletion previously reported, indicating this region could be a hot spot for gross SDHB deletions. It seems likely that these alterations can account for a considerable number of both familial and apparently sporadic paraganglioma cases. Although this is the first report describing the presence of gross deletions in patients with apparently sporadic paragangliomas, the extra‐adrenal location of the tumor seems to constitute a determining factor for whether to include these patients in genetic testing for gross deletions in the SDHB gene.