Flora Guerra

The PGC-1α-dependent pathway of mitochondrial biogenesis is upregulated in type I endometrial cancer

PGC-1alpha-dependent pathway of mitochondrial biogenesis was investigated for the first time in type I endometrial cancer and in normal endometrium. In cancer endometrial tissue the citrate synthase activity, the mitochondrial DNA content and the TFAM level were found doubled compared to control endometrial tissue. Moreover, a 1.6- and 1.8-fold increase, respectively, of NRF-1 and PGG-1alpha expression was found. This study demonstrates, for the first time, that the increased mitochondrial biogenesis in type I endometrial cancer is associated to the upregulation of PGC-1alpha signalling pathway.

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma.

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.

BRCA-Associated Ovarian Cancer: From Molecular Genetics to Risk Management

Ovarian cancer (OC) mostly arises sporadically, but a fraction of cases are associated with mutations in BRCA1 and BRCA2 genes. The presence of a BRCA mutation in OC patients has been suggested as a prognostic and predictive factor. In addition, the identification of asymptomatic carriers of such mutations offers an unprecedented opportunity for OC prevention. This review is aimed at exploring the current knowledge on epidemiological and molecular aspects of BRCA-associated OC predisposition, on pathology and clinical behavior of OC occurring in BRCA mutation carriers, and on the available options for managing asymptomatic carriers.

Mitochondrial DNA Mutation in Serous Ovarian Cancer: Implications for Mitochondria-Coded Genes in Chemoresistance

Case Report A69-year-oldwomansufferingdyspneaandwidespreadabdominal pain was admitted. Preliminary tests detected mild anemia, leukocytosis, and high levels of CA125 (2,200 U/mL). Abdominal-pelvic ultrasound scan, toraco-abdominal computer tomography (CT) and positron emission tomography (PET) indicated a gastric expansive mass together with two adherent pelvic solid masses, vascularized, strictly contiguous to bowel, omental cake, peritoneal carcinomatosis marks and ascites (Figs 1A and 1C; Figs 1E and 1G, arrows). Esophageal-gastric duodenoscopy was normal. CT-guided biopsy of the pelvic masses led to diagnose a poorly differentiated ovarian carcinoma in July 2010. A six-cycle carboplatin-paclitaxel chemotherapy was administered from August 2010 to December 2010. Radiological CT and PET at the end of the chemotherapyshowednearlycomplete response(Figs1Band1D;1Fand 1H). Diagnostic laparoscopy confirmed the absence of peritoneal carcinomatosis and suggested that surgery had become feasible. In March 2011, the patient was submitted to hysterectomy with bilateral annessiectomy, pelvic and lomboartic lymphadenectomy, omentectomy and peritoneal biopsies with complete macroscopical removal of disease. The histologicalreportshowedpersistenceofmicroscopicfociofdisease inthe ovaries (G3serouspapillaryadenocarcinoma),with lymphnodes,omentum and peritoneum free from disease. No adjuvant chemotherapy was administered because the patient had received six full courses of preoperativechemotherapyandnomacroscopicdiseasewaspresentaftersurgery. The patient was enrolled in the Mitochondria in Progression of Endometrial and Ovarian Cancer (MiPEO) study approved by the local ethical committee at S. Orsola Hospital, Bologna. Signed informed consent was obtained. She was subjected to regular follow-ups every 4 months with CT, pelvic ultrasound, and test of CA125. With the aim to characterize the residual chemoresistant ovarian cancer, hematoxylin/eosin staining showed a clear-cut oncocytic component, undetected in the prechemotherapy biopsy. Therefore, the acquisition of oncocytic change was observed exclusively in the ovarian cancer tissueresidualafterchemotherapy(Fig2).Wehavepreviouslyshownthat mitochondrial DNA (mtDNA) mutations in respiratory complex I (CI) genes are markers of oncocytic transformation, which cause CI derangement and, consequently, impairment of mitochondrial respiration. In order to understand whether mtDNA mutations and CI disassembly underlay the oncocytic phenotype, whole mtDNA sequencing was performed. Sequence analysis revealed the presence of m.10875T C missense mutation of a conserved residue (p.39Leu Pro) in the MTND4 gene encoding a CI subunit (Figs 3 and 4 [boxed in red in Fig 4]). The mutation was nearly homoplasmic, although a minor contamination withadjacentnontumortissuecouldnotbecompletelyruledout.Predictor of amino acid change pathogenic potential PolyPhen2 revealed the mutation to be probably damaging. Proof of pathogenicity was obtained through negative immunohistochemistry (IHC) staining of nuclearencoded CI subunit NDUFB8, which does not integrate within the complex when ND4 is lacking (Fig 2). In order to ascertain that the mtDNA disruptive mutation had accumulated postchemotherapy, normal and prechemotherapy tissues were screened for the same mutation, which was shown to be absent (Fig 3). In order to exclude the presence of a possible low-level heteroplasmy, a sensitive locked nucleic acid–based technique with a detection limit of 0.1% was implemented. Green squares represent standard curve samples (slope: 3.364; r 0.995; PCR efficiency: 97%). Wild-type control is circled in black and prechemotherapy sample in red. Absence of the mutation in prechemotherapy and in nontumor tissue was confirmed (Fig 5), indicating that the homoplasmic m.10875T Cwasapostchemotherapy-specificevent,consistentwiththe NDUFB8-positive staining of prechemotherapy tissue (Fig 2). Concordantly with the previously determined correlation between the occurrence of mtDNA mutations and low proliferation of oncocytic tumors, Ki67 of the postchemotherapy mass was nearly zero (Fig 6). Next, we sought to determine whether the residual mass had retained the original pro-oncogenic lesions, in order to ascertain a modifier role for the MTND4 mutation in keeping the chemoresistant clone under alow-proliferativeconstraint.Screeningofoncogenesfrequentlyinvolved in ovarian carcinogenesis, namely KRAS, BRAF, CTNNB1, PIK3CA, ERBB2 and AKT1 did not reveal mutations. Screening of tumor suppressor genes TP53 and PTEN identified a TP53 pathogenic heterozygous p.220Y C mutation both in preand postchemotherapic tissue, but not in the nontumor lymph node (Fig 7). Moreover, the homozygous TP53 p.72P R variant detected in all three samples (Fig 7) was previously reported to render TP53 a sensitive target for oncogenic protein E6 of human papilloma virus strain 16 (HPV16), occasionally associated with ovarian carcinoma. HPV16 was indeed detected both in tumor and in thenontumorlymphnodeofthepatientas it isevidentfromalignmentof detected amino acidic viral sequence (Fig 8). These data suggested cell transformation was due to the viral oncogenic properties in combination with the TP53 mutations.

Mitochondrial DNA content and mass increase in progression from normal to hyperplastic to cancer endometrium

BackgroundAn increase in mitochondrial DNA (mtDNA) content and mitochondrial biogenesis associated with the activation of PGC-1α signalling pathway was previously reported in type I endometrial cancer. The aim of this study has been to evaluate if mtDNA content and the citrate synthase (CS) activity, an enzyme marker of mitochondrial mass, increase in progression from control endometrium to hyperplasia to type I endometrial carcinoma.ResultsGiven that no statistically significant change in mtDNA content and CS activity in endometrium taken from different phases of the menstrual cycle or in menopause was found, these samples were used as control. Our research shows, for the first time, that mtDNA content and citrate synthase activity increase in hyperplastic endometrium compared to control tissues, even if their levels remain lower compared to cancer tissue. In particular, mtDNA content increases seem to precede increases in CS activity. No statistically significant change in mtDNA content and in CS activity was found in relation to different histopathological conditions such as grade, myometrial invasion and stage.ConclusionMtDNA content and citrate synthase activity increases in pre-malignant lesions could be a potential molecular marker for progression from hyperplasia to carcinoma.

Mitochondrial DNA depletion sensitizes cancer cells to PARP inhibitors by translational and post-translational repression of BRCA2

Previous studies have shown that pharmacologic inhibition of poly (ADP-ribose) polymerase (PARP), a nuclear protein that is crucial in signaling single-strand DNA breaks, is synthetically lethal to cancer cells from patients with genetic deficiency in the DNA repair proteins BRCA1 and BRCA2. Herein, we demonstrate that depletion of the mitochondrial genome (mtDNA) in breast, prostate and thyroid transformed cells resulted in elevated steady-state cytosolic calcium concentration and activation of calcineurin/PI3-kinase/AKT signaling leading to upregulation of miR-1245 and the ubiquitin ligase Skp2, two potent negative regulators of the tumor suppressor protein BRCA2, thus resulting in BRCA2 protein depletion, severe reduction in homologous recombination (HR) and increased sensitivity to the PARP inhibitor rucaparib. Treatment of mtDNA-depleted cells with the PI3-kinase inhibitor LY294002, the calmodulin antagonist W-7, the calcineurin inhibitor FK506, the calcium chelator BAPTA-AM, or suppression of AKT activity by AKT small-interfering RNA (siRNA) enhanced BRCA2 protein levels as well as HR. Decreasing the intracellular calcium levels using BAPTA, or direct reconstitution of BRCA2 protein levels either by recombinant expression or by small molecule inhibition of both Skp2 and miR-1245 restored sensitivity to rucaparib to wild-type levels. Furthermore, by studying prostate tissue specimens from prostate carcinoma patients we found a direct correlation between the presence of mtDNA large deletions and loss of BRCA2 protein in vivo, suggesting that mtDNA status may serve as a marker to predict therapeutic efficacy to PARP inhibitors. In summary, our results uncover a novel mechanism by which mtDNA depletion restrains HR, and highlight the role of mtDNA in regulating sensitivity to PARP inhibitors in transformed cells.

Mitochondrial DNA genotyping efficiently reveals clonality of synchronous endometrial and ovarian cancers.

Simultaneous independent primary tumors of the female genital tract occur in 1–2% of gynecological cancer patients, 50–70% of which are synchronous tumors of the endometrium and ovary. Recognition of synchrony upon multiple tumors is crucial for correct prognosis, therapeutic choice, and patient management. Current guidelines for determining synchrony, based on surgical and histopathological findings, are often ambiguous and may require further molecular analyses. However, because of the uniqueness of each tumor and of its intrinsic heterogeneity, these analyses may sometimes be inconclusive. A role for mitochondrial DNA genotyping was previously demonstrated in the diagnosis of synchronous endometrial and ovarian carcinoma. We have analyzed 11 sample pairs of simultaneously revealed endometrial and ovarian cancers and have thereby applied conventional histopathological criteria, current molecular analyses (microsatellite instability, β-catenin immunohistochemical staining/CTNNB1 mutation screening), and mitochondrial DNA sequencing to distinguish separate independent tumors from metastases, comparing the performance and the informative potential of such methods. We have demonstrated that in ambiguous interpretations where histopathological criteria and canonical molecular methods fail to be conclusive, mitochondrial DNA analysis may act as a needle of balance and allow to formulate a diagnosis in 45.5% of our cases. Additional advantages of mitochondrial DNA genotyping, besides the high level of information we demonstrated here, are the easy implementation and the need for small amounts of starting material. Our results show that mitochondrial DNA genotyping may provide a substantial contribution to indisputably recognize the metastatic nature of simultaneously detected endometrial and ovarian cancers and may change the final staging and clinical management of these patients.

Where Birt–Hogg–Dubé meets Cowden Syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours

Birt–Hogg–Dubè (BHD) is an autosomal dominant syndrome characterised by skin fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal cancer. The association of benign cutaneous lesions and increased cancer risk is also a feature of Cowden Syndrome (CS), an autosomal dominant disease caused by PTEN mutations. BHD and CS patients may develop oncocytomas, rare neoplasias that are phenotypically characterised by a prominent mitochondrial hyperplasia. We here describe the genetic analysis of a parotid and a thyroid oncocytoma, developed by a BHD and a CS patient, respectively. The BHD lesion was shown to maintain the wild-type allele of FLCN, while losing one PTEN allele. On the other hand, a double heterozygosity for the same two genes was found to be the only detectable tumorigenic hit in the CS oncocytoma. Both conditions occurred in a context of high chromosomal stability, as highlighted by comparative genomic hybridisation analysis. We conclude that, similarly to PTEN, FLCN may not always follow the classical Two Hits model of tumorigenesis and may hence belong to a class of non-canonical tumour suppressor genes. We hence introduce a role of PTEN/FLCN double heterozygosity in syndromic oncocytic tumorigenesis, suggesting this to be an alternative determinant to pathogenic mitochondrial DNA mutations, which are instead the genetic hallmark of sporadic oncocytic tumours.

Mitochondrial DNA genotyping reveals synchronous nature of simultaneously detected endometrial and ovarian cancers.

Simultaneous independent primary tumors of the female genital tract occur in 1-2% of gynecological cancer patients, 50-70% of which are synchronous tumors of the endometrium and ovary. Guidelines for determining the nature of simultaneously detected tumors, based on surgical and histopathological findings, are often ambiguous and may require further molecular analyses. Such approach is necessary to indicate correct prognosis and hence treatment. We here demonstrate how mitochondrial DNA sequencing may provide a cheap and useful tool to contribute to indisputably recognize the synchronous nature of simultaneously detected endometrial and ovarian carcinomas. We further confirm our findings by means of Comparative Genomic Hybridization array analysis, which strengthens the informative potential of mitochondrial DNA genotyping in diagnosing synchrony.

Metabolites from invasive pests inhibit mitochondrial complex II: A potential strategy for the treatment of human ovarian carcinoma?

The red pigment caulerpin, a secondary metabolite from the marine invasive green algae Caulerpa cylindracea can be accumulated and transferred along the trophic chain, with detrimental consequences on biodiversity and ecosystem functioning. Despite increasing research efforts to understand how caulerpin modifies fish physiology, little is known on the effects of algal metabolites on mammalian cells. Here we report for the first time the mitochondrial targeting activity of both caulerpin, and its closely related derivative caulerpinic acid, by using as experimental model rat liver mitochondria, a system in which bioenergetics mechanisms are not altered. Mitochondrial function was tested by polarographic and spectrophotometric methods. Both compounds were found to selectively inhibit respiratory complex II activity, while complexes I, III, and IV remained functional. These results led us to hypothesize that both algal metabolites could be used as antitumor agents in cell lines with defects in mitochondrial complex I. Ovarian cancer cisplatin-resistant cells are a good example of cell lines with a defective complex I function on which these molecules seem to have a toxic effect on proliferation. This provided novel insight toward the potential use of metabolites from invasive Caulerpa species for the treatment of human ovarian carcinoma cisplatin-resistant cells.