Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P ؍ 0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.oncocytic tumors ͉ heteroplasmy ͉ homoplasmy ͉ damaging mutation ͉ microenvironment
The oncogenic versus suppressor roles of mitochondrial genes have long been debated. Peculiar features of mitochondrial genetics such as hetero/homoplasmy and mutation threshold are seldom taken into account in this debate. Mitochondrial DNA (mtDNA) mutations generally have been claimed to be protumorigenic, but they are also hallmarks of mostly benign oncocytic tumors wherein they help reduce adaptation to hypoxia by destabilizing hypoxia-inducible factor-1a (HIF1a). To determine the influence of a disassembling mtDNA mutation and its hetero/homoplasmy on tumorigenic and metastatic potential, we injected mice with tumor cells harboring different loads of the gene MTND1 m.3571insC. Cell cultures obtained from tumor xenografts were then analyzed to correlate energetic competence, apoptosis, a-ketoglutarate (a-KG)/succinate (SA) ratio, and HIF1a stabilization with the mutation load. A threshold level for the antitumorigenic effect of MTND1 m.3571insC mutation was defined, above which tumor growth and invasiveness were reduced significantly. Notably, HIF1a destabilization and downregulation of HIF1a-dependent genes occurred in cells and tumors lacking complex I (CI), where there was an associated imbalance of a-KG/SA despite the presence of an actual hypoxic environment. These results strongly implicate mtDNA mutations as a cause of oncocytic transformation. Thus, the antitumorigenic and antimetastatic effects of high loads of MTND1 m.3571insC, following CI disassembly, define a novel threshold-regulated class of cancer genes. We suggest these genes be termed oncojanus genes to recognize their ability to contribute either oncogenic or suppressive functions in mitochondrial settings during tumorigenesis.
BackgroundAerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression.MethodsComplex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts.ResultsComplementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue.ConclusionRespiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.
The present paper documents an investigation of the morphology, immunohistochemistry, and ultrastructure of Toker cells (TC), aiming for a better definition of these elements and better understanding of their histogenesis. We studied 12 nipples removed for nipple adenoma from twelve patients and a case of supernumerary nipple. In addition four cases of Paget's carcinoma (PC) restricted to the nipple without underlying tumor were studied for comparison. All cases were stained with hematoxylin and eosin (H&E), Alcian blue pH 2.5 and periodic acid-Schiff (PAS) preceded by diastase digestion and with immunohistochemistry using antisera anti cytokeratin 7, cytokeratin 20, protein S100, GCDFP-15, c-Erb-B2, CAM 5.2, and epithelial membrane antigen (EMA). Two cases from the nipple adenoma series were studied by electron microscopy. In seven cases within the series of 12 nipple adenomas as well as in the case of supernumerary nipple, keratin 7 antibody highlighted numerous cells located within the nipple epidermis which in three cases showed dendritic processes. These same elements were also positive with CAM 5.2. All these same elements were negative with Alcian Blue (AB), PAS and the other antisera employed. Ultrastructural examination demonstrated that these cells differed from keratinocytes while they presented the same features as the glandular cells seen in the related nipple adenoma. The cells constituting Paget's carcinoma showed more irregular nuclei and were more easily seen in the context of the epidermis. The immunocytochemical profile of the cancer cells was similar to that of TC, but in addition the neoplastic cells were c-Erb-B2 and EMA positive in all cases, and one case also displayed numerous cells immunoreactive with anti GCDFP-15 antibody. Keratin 7 highlighted dendritic cells in two cases and AB, PAS was negative in all patients. The immunocytochemical profile and the ultrastructural features of TC are similar to those of the glandular cells constituting the ducts and the adenoma. These findings together with the localization of TC near or around the openings of the lactiferous sinuses indicate that TC might be ductal cells with a dendritic aspect and migrate through the galactophorous ostia. PC cells not related to ductal carcinomas have a similar but not superimposable immunohistochemical profile to TC, and in two cases the neoplastic elements were also dendritic which suggests that these same cells are likely to be the neoplastic counterpart of TC.
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