David Petillo

Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes

Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer, characterized by the presence of inactivating mutations in the VHL gene in most cases, and by infrequent somatic mutations in known cancer genes. To determine further the genetics of ccRCC, we have sequenced 101 cases through 3,544 protein-coding genes. Here we report the identification of inactivating mutations in two genes encoding enzymes involved in histone modification—SETD2, a histone H3 lysine 36 methyltransferase, and JARID1C (also known as KDM5C), a histone H3 lysine 4 demethylase—as well as mutations in the histone H3 lysine 27 demethylase, UTX (KMD6A), that we recently reported. The results highlight the role of mutations in components of the chromatin modification machinery in human cancer. Furthermore, NF2 mutations were found in non-VHL mutated ccRCC, and several other probable cancer genes were identified. These results indicate that substantial genetic heterogeneity exists in a cancer type dominated by mutations in a single gene, and that systematic screens will be key to fully determining the somatic genetic architecture of cancer.

HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome.

We report here the identification of a gene associated with the hyperparathyroidism–jaw tumor (HPT–JT) syndrome. A single locus associated with HPT–JT (HRPT2) was previously mapped to chromosomal region 1q25–q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT–JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT–JT and in development of some sporadic parathyroid tumors.

Interleukin-8 Mediates Resistance to Antiangiogenic Agent Sunitinib in Renal Cell Carcinoma

The broad spectrum kinase inhibitor sunitinib is a first-line therapy for advanced clear cell renal cell carcinoma (ccRCC), a deadly form of kidney cancer. Unfortunately, most patients develop sunitinib resistance and progressive disease after about 1 year of treatment. In this study, we evaluated the mechanisms of resistance to sunitinib to identify the potential tactics to overcome it. Xenograft models were generated that mimicked clinical resistance to sunitinib. Higher microvessel density was found in sunitinib-resistant tumors, indicating that an escape from antiangiogenesis occurred. Notably, escape coincided with increased secretion of interleukin-8 (IL-8) from tumors into the plasma, and coadministration of an IL-8 neutralizing antibody resensitized tumors to sunitinib treatment. In patients who were refractory to sunitinib treatment, IL-8 expression was elevated in ccRCC tumors, supporting the concept that IL-8 levels might predict clinical response to sunitinib. Our results reveal IL-8 as an important contributor to sunitinib resistance in ccRCC and a candidate therapeutic target to reverse acquired or intrinsic resistance to sunitinib in this malignancy.

HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours

Background: Hyperparathyroidism is a common endocrinopathy characterised by the formation of parathyroid tumours. In this study, we determine the role of the recently identified gene, HRPT2, in parathyroid tumorigenesis. Methods: Mutation analysis of HRPT2 was undertaken in 60 parathyroid tumours: five HPT-JT, three FIHP, three MEN 1, one MEN 2A, 25 sporadic adenomas, 17 hyperplastic glands, two lithium associated tumours, and four sporadic carcinomas. Loss of heterozygosity at 1q24-32 was performed on a subset of these tumours. Results:HRPT2 somatic mutations were detected in four of four sporadic parathyroid carcinoma samples, and germline mutations were found in five of five HPT-JT parathyroid tumours (two families) and two parathyroid tumours from one FIHP family. One HPT-JT tumour with germline mutation also harboured a somatic mutation. In total, seven novel and one previously reported mutation were identified. “Two-hits” (double mutations or one mutation and loss of heterozygosity at 1q24-32) affecting HRPT2 were found in two sporadic carcinomas, two HPT-JT-related and two FIHP related tumours. Conclusions: The results in this study support the role of HRPT2 as a tumour suppressor gene in sporadic parathyroid carcinoma, and provide further evidence for HRPT2 as the causative gene in HPT-JT, and a subset of FIHP. In light of the strong association between mutations of HRPT2 and sporadic parathyroid carcinoma demonstrated in this study, it is hypothesised that HRPT2 mutation is an early event that may lead to parathyroid malignancy and suggest intragenic mutation of HRPT2 as a marker of malignant potential in both familial and sporadic parathyroid tumours.

Preparing the “Soil”: The Primary Tumor Induces Vasculature Reorganization in the Sentinel Lymph Node before the Arrival of Metastatic Cancer Cells

Sentinel lymph node (SLN) metastasis is the first step in the spreading of cancer in many malignancies. Tumor-reactive lymphadenopathy in SLNs has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain unexplored. Using animal models, we show here that, before the establishment of metastasis in the SLN, there are reorganizations of the lymphatic channels and the vasculature. The node becomes a functional blood vessel-enriched and lymph vessel/sinus-enriched organ before metastasis. The enlargement of the lymph sinuses is correlated with the primary tumor weight. The newly emerged functional blood vessels develop from high endothelial venules (HEV), in which the proliferation rate of the endothelial cells is also significantly increased. Similar alterations of the HEVs are also characterized in the axillary lymph nodes from human breast cancer patients without the evidence of metastasis. These findings support the hypothesis that modification of the microenvironment for a secondary tumor (i.e., vasculature reorganization in the SLN) can be initiated by a primary tumor before and independent of the physical presence of metastatic cancer cells.

Renal translocation carcinomas: clinicopathologic, immunohistochemical, and gene expression profiling analysis of 31 cases with a review of the literature.

We report clinicopathologic features of a large series of renal translocation carcinomas from a multicentric study. Diagnosis was performed by cytogenetic examination of fresh material and/or by immunochemistry with antibodies directed against the C-terminal part of transcription factor E3 (TFE3) and native transcription factor EB (TFEB) proteins. Clinical data, follow-up, and histologic features were assessed. Antibodies against CK7, CD10, vimentin, epithelial membrane antigen, AE1-AE3, E-cadherin, α-methylacyl-coenzyme A racemase, melan A, and HMB45 were tested on tissue microarrays. Whole-genome microarray expression profiling was performed on 4 tumors. Twenty-nine cases were diagnosed as TFE3 and 2 as TFEB renal translocation carcinomas, including 13 males and 18 females, mean age 24.6 years. Two patients had a previous history of chemotherapy and 1 had a history of renal failure. Mean size of the tumor was 6.9 cm. Thirteen cases were ≥pT3 stage. Twelve cases were N+ or M+. Mean follow-up was 29.5 months. Three patients presented metastases and 5 have died. Mixed papillary and nested patterns with clear and/or eosinophilic cells represented the most consistent histologic appearance, with common foci of calcifications regardless of the type of translocation. Using a 30 mn incubation at room temperature, TFE3 immunostainings were positive in only 82% of our TFE3 translocation carcinomas. Both TFE3 and TFEB renal translocation carcinomas expressed CD10 and α-methylacyl-coenzyme A racemase in all cases. An expression of E-cadherin was observed in two-third of cases. Cytokeratins were expressed in less than one-third of cases. Melanocytic markers were expressed at least weakly in all cases except two. Unsupervised clustering on the basis of the gene expression profiling indicated a distinct subgroup of tumors. TRIM 63 glutathione S-transferase A1 and alanyl aminopeptidase are the main differentially expressed genes for this group of tumors. Our results suggest that these differentially expressed genes may serve as novel diagnostic or prognostic markers.

Deficiency of FLCN in Mouse Kidney Led to Development of Polycystic Kidneys and Renal Neoplasia

The Birt–Hogg–Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHDflox/flox/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHDflox/flox/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD flox/+/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome–related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.

The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas

By positional cloning, we identified two breakpoint-spanning genes in a familial clear cell renal cell carcinoma (CCRCC)-associated t(1;3)(q32.1;q13.3): LSAMP and NORE1 (RASSF1 homolog). Both genes are downregulated in 9 of 9 RCC cell lines. While the NORE1A promoter predominantly presents partial methylation in 6 of the cell lines and 17/53 (32%) primary tumors, the LSAMP promoter is completely methylated in 5 of 9 cell lines and in 14/53 (26%) sporadic and 4 familial CCRCCs. Expression of LSAMP and NORE1A proteins in CCRCC cell lines inhibited cell proliferation. These characteristics indicate that LSAMP and NORE1A may represent new candidate tumor suppressors for CCRCC.

Germline and de novo mutations in the HRPT2 tumour suppressor gene in familial isolated hyperparathyroidism (FIHP)

Familial forms of primary hyperparathyroidism (PHPT) constitute a broad group of disorders in which PHPT is either a main or an associated feature. With the advances in disease gene identification, some of the genetic abnormalities underlying familial PHPT have been clarified.1,2 In hyperparathyroidism–jaw tumour syndrome (HPT-JT; OMIM #145001) the affected family members frequently develop PHPT, ossifying jaw fibromas, and cystic and neoplastic renal lesions.3–6 A typical feature of HPT-JT is adenomas and carcinomas of the parathyroid glands, which often have cystic features.1 This is in contrast to the other forms of familial PHPT in which the parathyroid tumours are generally benign. The disease locus was first mapped to chromosomal region 1q25–q32 by linkage in affected families3,5–7 and recently the causal HRPT2 gene was isolated through a positional cloning approach.3 The HRPT2 gene consists of 17 exons encoding an evolutionarily well conserved, 531 amino acid protein named parafibromin. The inactivating mutations demonstrated in the germline of HPT-JT kindreds and as somatic events in some sporadic parathyroid adenomas3 are in agreement with the observations of somatic loss of the wild type alleles,6 suggesting that parafibromin has a tumour suppressor function.3,6 The importance of the multiple endocrine neoplasia type 1 gene ( MEN1 ) in familial PHPT has been well established. MEN1 is a tumour suppressor gene located in 11q13,8–10 and its encoded protein menin has been shown to interact with several proteins involved in transcriptional regulation.11,12 The MEN1 syndrome (OMIM #131100) is clinically characterised by the frequent development of tumours in the parathyroids, the endocrine pancreas and duodenum, and the anterior pituitary gland. MEN1, which is the most common form of hereditary PHPT, is caused by germline mutations of MEN1 , both in the form of inherited …

Serglycin Is a Theranostic Target in Nasopharyngeal Carcinoma that Promotes Metastasis

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial-mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients.