Ana S. Pitiot

Loss of collagenase-2 confers increased skin tumor susceptibility to male mice

Matrix metalloproteinases (MMPs) have fundamental roles in tumor progression, but most clinical trials with MMP inhibitors have not shown improvements in individuals with cancer. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8−/−mice. Female Mmp8−/−mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.

Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8)

Matrix metalloproteinases (MMPs) have been implicated in numerous tissue‐remodeling processes. The finding that mice deficient in collagenase‐2 (MMP‐8) are more susceptible to develop skin cancer, prompted us to investigate the role of this protease in cutaneous wound healing. We have observed a significant delay in wound closure in MMP8−/− mice and an altered inflammatory response in their wounds, with a delay of neutrophil infiltration during the first days and a persistent inflammation at later time points. These changes were accompanied by alterations in the TGF‐β1 signaling pathway and by an apoptosis defect in MMP8−/− mice. The delay in wound healing observed in MMP8−/− mice was rescued by bone marrow transplantation from wild‐type mice. Analysis of other MMPs showed that MMP8−/−mice had a significant increase in the expression of MMP‐9, suggesting that both proteases might act coordi‐nately in this process. This possibility was further supported by the novel finding that MMP‐8 and MMP‐9 form specific complexes in vivo. Taken together, these data indicate that MMP‐8 participates in wound repair by contributing to the resolution of inflammation and open the possibility to develop new strategies for treating wound healing defects.—Gutierrez‐Fernandez, A., Inada, M., Balbín, M., Fueyo, A., Pitiot, A. S., Astudillo, A., Hirose, K., Hirata, M., Shapiro, S. D., Noel, A., Werb, Z., Krane, S. M. Lopez‐Otín, C., Puente, X. S. FASEB J. 21, 2580–2591 (2007)

Mutational analysis of BRCA1 and BRCA2 in hereditary breast and ovarian cancer families from Asturias (Northern Spain)

BackgroundThe prevalence of BRCA1 and BRCA2 mutations in Spain is heterogeneous and varies according to geographical origin of studied families. The contribution of these mutations to hereditary breast and ovarian cancer has not been previously investigated in Asturian populations (Northern Spain).MethodsIn the present work, 256 unrelated high-risk probands with breast and/or ovarian cancer from families living in Asturias were analyzed for the presence of a BRCA1 or BRCA2 gene mutation from October 2007 to May 2012. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened both by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA).ResultsA total of 59 families (23%) were found to carry a pathogenic germ line mutation, 39 in BRCA1 and 20 in BRCA2. Twenty nine additional families (12%) carried an unknown significance variant. We detected 28 distinct pathogenic mutations (16 in BRCA1 and 12 in BRCA2), of which 3 mutations in BRCA1 (c.1674delA, c.1965C>A and c.2900_2901dupCT) and 5 in BRCA2 (c.262_263delCT, c.2095C>T, c.3263dupC, c.4030_4035delinsC, c.8042_8043delCA) had not been previously described.The novel mutations c.2900_2901dupCT in BRCA1 and c.4030_4035delinsC in BRCA2 occurred in 8 and 6 families respectively and clustered in two separated small geographically isolated areas suggesting a founder effect. These 2 mutations, together with the Galician BRCA1 mutation c.211A>G (9 families), and the common BRCA1 mutation c.3331_3334delCAAG (6 families), account for approximately 50% of all affected families. By contrast, very frequent mutations in other Spanish series such as the BRCA1 Ashkenazi founder mutation c.68_69delAG, was found in only one family.ConclusionsIn this study we report the BRCA1 and BRCA2 spectrum of mutations and their geographical distribution in Asturias, which largely differ from other areas of Spain. Our findings may help design a first step recurrent mutation panel for screening high-risk breast and/or ovarian cancer families from this specific area.

Germ-line mutations in epidermal growth factor receptor (EGFR) are rare but may contribute to oncogenesis: A novel germ-line mutation in EGFR detected in a patient with lung adenocarcinoma

BackgroundA subset of lung cancer patients harbour EGFR somatic mutations in their tumours and are candidates for treatment with EGFR tyrosine kinase inhibitors. In a few cases EGFR mutations have also been found in the germ line, suggesting a role in lung carcinogenesis. Objetives of this study were: 1) To analyze the EGFR gene mutations in a population diagnosed with lung adenocarcinoma from Northern Spain. 2) To determine the frequency of a new germ-line mutation found in our laboratory as well as the frequency in our population of three other EGFR germ-line mutations detected by other authors. 3) To determine whether the novel mutation detected may have a functional effect on the EGFR protein.MethodsTumour DNA samples were obtained from frozen or paraffin embedded tumour tissues. Samples of DNA from peripheral blood cells were obtained from 912 individuals with lung cancer recruited from the CAPUA study [1, 2], 477 unrelated healthy donor individuals and 32 individuals with other types of cancer. EGFR gene exons 18 to 21 were studied by direct standard dideoxy sequencing. Specific mutations were determined either by direct sequencing or by specific RFLP analysis. Cell lines were transfected with EGFR-mutant plasmids and analysed by western blot with antibodies specific for total or phosphorylated-EGFR.ResultsWe found EGFR mutation in 12 of the 71 tumour samples (17%). One tumour contained two mutations. One mutation (p.R776G) was present as a germ line. Using an RFLP analysis, this mutation was not found in 954 alleles from healthy individuals studied, concluding that it is not a polymorphism. The mutation was not found either in genomic DNA from 912 lung cancer patients. Three additional EGFR germ-line mutations that were already described were not found in any of the studied samples. These observations show that EGFR mutated alleles are rare in the population. In vitro studies revealed that tyrosine autophosphorylation is enhanced in p.R776G-mutant EGFR when compared with wild-type EGFR. This enhanced autophosphorylation in the absence of ligand may be associated with a proliferative advantage.ConclusionsGerm-line mutations in EGFR are rare but may contribute to oncogenesis

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

CONTEXT Head 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). OBJECTIVE The present study aimed at unraveling the SDH-independent mechanisms involved in the activation of HIF in HNPGLs. DESIGN The 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. RESULTS We 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. CONCLUSIONS VHL, 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.

ABL alternative splicing is quite frequent in normal population - letter.

In the article by Lee et al. (1), an alternative splicing of BCR-ABL is proposed as a mechanism for imatinib resistance in chronic myeloid leukemia (CML) patients. This splicing (2), which inserts 35 bp between exons 8 and 9 of ABL (35INS), results in a truncated BCR-ABL protein that the authors compare dynamically with the native protein. We found their work very relevant, especially the finding on the conformational change and its similarity with tertiary structures due to resistance mutations. We were nonetheless surprised by the high proportion of patients who became resistant to imatinib with the alternative splicing; however, the authors did not present data on the presence of the 35-bp insertion in other populations or native ABL mRNA. Using a similar experimental approach to the one described in their article, we analyzed 18 samples from CML patients treated with kinase inhibitors and 24 samples from a normal population. The analysis of the normal population may serve as an estimation of the alternative splicing frequency of the ABL gene. The results showed that 27% of the patients with CML and 41.6% of the normal population expressed 35INS in different degrees (only those with >5% alternatively spliced were considered positive, as described by Lee et al.). In the samples from CML patients, we were unable to correlate either the presence or relative levels of expression of INS35 to tyrosine kinase inhibitor resistance. Several patients who had suboptimal responses to imatinib and an absence of ABL mutations were negative for INS35. One patient, who was marginally positive (5.1%) for INS35 and have a F359V mutation, presented a BCR-ABL / GUS ratio of 22.3%; after being switched to dasatinib treatment, his BCR-ABL ratio decreased to 0.08% in 4 months, but showed an INS35 presence of 24%, which indicates that the splicing was occurring in nonmutated Ph+ cells. The BCR-ABL ratio of the patient, however, further decreased to undetectable levels in the following 4 months. Another patient, who failed to achieve a complete molecular response, was negative for INS35 or ABL mutation at that point; previous samples showed the presence of the alternative splicing in 48.4% of the BCR-ABL mRNA, which indicates that the splicing has decreased over time while the resistance has endured. All these data seem to confirm that INS35 may just be a pseudo-exon not uncommon in the ABL gene processing (3) and therefore unrelated to resistance or sensitivity (4) to inhibitors in CML treatment. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Identification of somatic and germ-line DICER1 mutations in pleuropulmonary blastoma, cystic nephroma and rhabdomyosarcoma tumors within a DICER1 syndrome pedigree

BackgroundDICER1 syndrome is a pediatric cancer predisposition condition causing a variety of tumor types in children and young adults. In this report we studied a family with two relatives presenting a variety of neoplastic conditions at childhood.MethodsGerm-line mutation screening of the complete coding region of the DICER1 gene in genomic DNA from the proband was performed. The presence of somatic DICER1 mutation and further alterations in driver genes was investigated in genomic DNA obtained from available tumor samples.ResultsA nonsense germ-line mutation in DICER1 causing a truncated protein at the IIIb domain level was identified segregating within a family including two affected relatives who developed in one case cystic nephroma and pleuropulmonary blastoma, and rhabdomyosarcoma and multinodular goiter in the other. Additional in trans DICER1 missense somatic mutations in the IIIb DICER1 domain were found both in the cystic nephroma and in the rhabdomyosarcoma, suggesting that neoplasms in this family might arise from the unusual two-hit mechanism for DICER-derived tumorigenesis in which after the presence of a truncated constitutive protein, a neomorphic DICER1 activity is somatically adquired. Additional genetic alterations, such as TP53 mutations, were identified in the rhabdomyosarcoma.ConclusionsBesides DICER1 loss of standard activity, oncogenic cooperation of other genes, as mutated TP53, may involve developing higher grade tumors within this syndrome. Given the broad clinical spectrum that may arise, genetic counseling and close surveillance must be offered to all family members at risk of DICER1 syndrome.

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.

A t(1;9) translocation involving CSF3R as a novel mechanism in unclassifiable chronic myeloproliferative neoplasm

Atypical chronic myeloid leukemia (aCML), chronic neutrophilic leukemia (CNL), and unclassifiable chronic myeloproliferative neoplasm (MPN-U) constitute rare hematological neoplasms characterized by the abnormal expansion of granulocytes or neutrophils in bone marrow and peripheral blood in absence

TP53 p.R72P genotype is a marker of poor prognosis in lung cancer

BACKGROUND Lung cancer is a leading cause of death worldwide, with poor survival rates despite diagnostic and therapeutic advances. Markers are needed in order to improve clinical patient management and survival. TP53 is frequently involved in lung cancer development with polymorphic sites potentially having a role in it. This study aims to determine the value of codon 72 missense polymorphic variant genotyping, TP53 R72P, as a prognostic factor in NSCLC patients. METHODS One hundred and fifteen NSCLC samples from patients exposed to tobacco smoke and silica dust from Asturias (Northern Spain) were genotyped by direct sequencing. RESULTS Seventy-five percent tumour samples alleles coded for Arg. The R72P genotype was an independent predictor of lymph node status (HR = 3.6). The heterozygous genotype was associated to a reduced 5-year survival rate (28% vs 51% for homozygotes). Importantly, this result was specifically observed in these subsets of patients: those over 67 years, patients with silicosis, current smokers, patients with squamous cell carcinomas and, notably, with tumour free lymph nodes. CONCLUSION Our results indicate a remarkable application of R72P genotyping in the clinical setting: refine patient subclassification to identify those with an adverse clinical course despite tumour free lymph node status.