Cristina Rodríguez-Antona

Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.

Cytochrome P450 pharmacogenetics and cancer

The cytochromes P450 (CYPs) are key enzymes in cancer formation and cancer treatment. They mediate the metabolic activation of numerous precarcinogens and participate in the inactivation and activation of anticancer drugs. Since all CYPs that metabolize xenobiotics are polymorphic, much emphasis has been put on the investigation of a relationship between the distribution of specific variant CYP alleles and risk for different types of cancer, but a consistent view does not yet exist. This is to a great extent explained by the fact that the CYPs involved in activation of precarcinogens are in general not functionally polymorphic. This is in contrast to CYPs that are active in drug biotransformation where large interindividual differences in the capacity to metabolize therapeutic drugs are seen as a consequence of polymorphic alleles with altered function. This includes also some anticancer drugs like tamoxifen and cyclophosphamide metabolized by CYP2D6, CYP2C19 and CYP2B6. Some P450 forms are also selectively expressed in tumours, and this could provide a mechanism for drug resistance, but also future therapies using these enzymes as drug targets can be envisioned. This review gives an up-to-date description of our current knowledge in these areas.

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells

1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP- β : LAP and LIP, HNF-3 α, HNF-4 α, RXR- α) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.

Single nucleotide polymorphism associations with response and toxic effects in patients with advanced renal-cell carcinoma treated with first-line sunitinib: a multicentre, observational, prospective study

BACKGROUND Sunitinib is a tyrosine kinase inhibitor with proven efficacy in renal-cell carcinoma, but some patients do not respond or need dose reductions due to toxicity. Because there are no validated molecular predictors of response or toxicity to sunitinib, we aimed to identify genetic markers predictive of outcome and toxic effects. METHODS In our observational, prospective study we enrolled previously untreated adults (≥ 18 years) with clear-cell renal-cell carcinoma at 15 institutions in the Spanish Oncology Genitourinary Group in Spain. Patients received sunitinib according to local practice guidelines. We assessed RECIST response, progression-free survival (PFS), overall survival, and toxicity of sunitinib with 16 key polymorphisms in nine genes: VEGFR2 (rs2305948 and rs1870377), VEGFR3 (rs307826, rs448012, and rs307821), PDGFR-α (rs35597368), VEGF-A (rs2010963, rs699947, and rs1570360), IL8 (rs1126647), CYP3A4 (rs2740574), CYP3A5 (rs776746), ABCB1 (rs1045642, rs1128503, and rs2032582), and ABCB2 (rs2231142). We assessed associations with efficacy and toxicity by use of univariable and multivariable analyses (with clinical factors associated with outcomes as covariates). We adjusted for multiplicity using the Bonferroni method; p values of less than 0·0031 before adjustment were deemed to still be significant after adjustment. FINDINGS We enrolled 101 patients between Oct 10, 2007, and Dec 13, 2010. 95 of these patients were included in toxicity analyses and 89 in the efficacy analyses. Two VEGFR3 missense polymorphisms were associated with reduced PFS with sunitinib on multivariable analysis: rs307826 (hazard ratio [HR] per allele 3·57, 1·75-7·30; p(unadjusted)=0·00049, p(adjusted)=0·0079) and rs307821 (3·31, 1·64-6·68; p(unadjusted)=0·00085, p(adjusted)=0·014). The CYP3A5*1 (rs776746) high metabolising allele was associated in a multivariable analysis with an increased risk of dose reductions due to toxicity (HR per allele 3·75, 1·67-8·41; p(unadjusted)=0·0014, p(adjusted)=0·022). No other SNPs were associated with sunitinib response or toxicity. INTERPRETATION Polymorphisms in VEGFR3 and CYP3A5*1 might be able to define a subset of patients with renal-cell carcinoma with decreased sunitinib response and tolerability. If confirmed, these results should promote interventional studies testing alternative therapeutic approaches for patients with such variants. FUNDING Pfizer.

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.

Tumoral EPAS1 (HIF2A) mutations explain sporadic pheochromocytoma and paraganglioma in the absence of erythrocytosis

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are chromaffin-cell tumors that arise from the adrenal medulla and extra-adrenal paraganglia, respectively. The dysfunction of genes involved in the cellular response to hypoxia, such as VHL, EGL nine homolog 1, and the succinate dehydrogenase (SDH) genes, leads to a direct abrogation of hypoxia inducible factor (HIF) degradation, resulting in a pseudo-hypoxic state implicated in PCC/PGL development. Recently, somatic post-zygotic mutations in EPAS1 (HIF2A) have been found in patients with multiple PGLs and congenital erythrocytosis. We assessed 41 PCCs/PGLs for mutations in EPAS1 and herein describe the clinical, molecular and genetic characteristics of the 7 patients found to carry somatic EPAS1 mutations; 4 presented with multiple PGLs (3 of them also had congenital erythrocytosis), whereas 3 were single sporadic PCC/PGL cases. Gene expression analysis of EPAS1-mutated tumors revealed similar mRNA EPAS1 levels to those found in SDH-gene- and VHL-mutated cases and a significant up-regulation of two hypoxia-induced genes (PCSK6 and GNA14). Interestingly, single nucleotide polymorphism array analysis revealed an exclusive gain of chromosome 2p in three EPAS1-mutated tumors. Furthermore, multiplex-PCR screening for small rearrangements detected a specific EPAS1 gain in another EPAS1-mutated tumor and in three non-EPAS1-mutated cases. The finding that EPAS1 is involved in the sporadic presentation of the disease not only increases the percentage of PCCs/PGLs with known driver mutations, but also highlights the relevance of studying other hypoxia-related genes in apparently sporadic tumors. Finally, the detection of a specific copy number alteration affecting chromosome 2p in EPAS1-mutated tumors may guide the genetic diagnosis of patients with this disease.

Molecular genetics and epigenetics of the cytochrome P450 gene family and its relevance for cancer risk and treatment

The cytochromes P450 (CYPs) are very efficient catalysts of foreign compound metabolism and are responsible for the major part of metabolism of clinically important drugs. The enzymes are important in cancer since they (a) activate dietary and environmental components to ultimate carcinogens, (b) activate or inactivate drugs used for cancer treatment, and (c) are potential targets for anticancer therapy. The genes encoding the CYP enzymes active in drug metabolism are highly polymorphic, whereas those encoding metabolism of precarcinogens are relatively conserved. A vast amount of literature is present where investigators have tried to link genetic polymorphism in CYPs to cancer susceptibility, although not much conclusive data have hitherto been obtained, with exception of CYP2A6 polymorphism and tobacco induced cancer, to a great extent because of lack of important functional polymorphisms in the genes studied. With respect to anticancer treatment, the genetic CYP polymorphism is of greater importance, where treatment with tamoxifen, but also with cyclophosphamide and maybe thalidomide is influenced by CYP genetic variants. In the present review we present updates on CYP genetics, cancer risk and treatment and also epigenetic aspects of interindividual variability in CYP expression and the use of these enzymes as targets for cancer therapy. We conclude that the CYP polymorphism does not predict cancer susceptibility to any large extent but that this polymorphism might be an important factor for optimal cancer therapy using selected anticancer agents.