Sharifeh Mehrabi

Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages

Background A majority of primary ovarian neoplasms arise from cell surface epithelium of the ovaries. Although old age and a positive family history are associated risk factors, the etiology of the epithelial ovarian tumors is not completely understood. Additionally, knowledge of factors involved in the histogenesis of the various subtypes of this tumor as well as those factors that promote progression to advanced stages of ovarian malignancy are largely unknown. Current evidence suggests that mitochondrial alterations involved in cellular signaling pathways may be associated with tumorigenesis. Methods In this study, we determined the presence of polymorphisms and other sequence variants of mitochondrial DNA (mtDNA) in 102 epithelial ovarian tumors including 10 matched normal tissues that paired with some of the tumors. High-resolution restriction endonucleases and PCR-based sequencing were used to assess the mtDNA variants spanning 3.3 kb fragment that comprised the D-Loop and 12S rRNA-tRNAphe, tRNAval, tRNAser, tRNAasp, tRNAlys, ATPase 6, ATPase 8, cytochrome oxidase I and II genes. Results Three hundred and fifty-two (352) mtDNA sequence variants were identified, of which 238 of 352 (68%) have not been previously reported. There were relatively high frequencies of three mutations in the 12S rRNA gene at np 772, 773, and 780 in stage IIIC endometrioid tumors, two of which are novel (773delT and 780delC), and occurred with a frequency of 100% (7/7). Furthermore, two mutations were observed in serous tumors only at np 1657 in stage IV (10/10), and at np 8221delA in benign cystadenomas (3/3) and borderline tumors (4/4). A high frequency, 81% (13/16) of TC insertion at np 310 was found only in early stages of serous subtype (benign cystadenomas, 3/3; borderline tumors, 4/4; stage I tumors, 2/5 and matched normal tissues 4/4). Conclusion Our findings indicate that certain mtDNA mutations can reliably distinguish the different histologic subtypes of epithelial ovarian tumors. In addition, these data raise the possibility that certain mtDNA mutations may be useful biomarkers for predicting tumor aggressiveness and may play a potential role in tumorigenesis.

Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro

Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson’s theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.

mtDNA sequence variants in subtypes of epithelial ovarian cancer stages in relation to ethnic and age difference

Epithelial ovarian cancer is the fifth leading cause of cancer mortality among women in the United States. For this disease, differences in age-adjusted incidence and survival rates between African American and Caucasian women are substantial. The objective of this study was to examine mtDNA sequence variants in 118 frozen tissues of three subtypes of epithelial ovarian cancer (serous, n = 48 endometrioid, n = 47 and mucinous, n = 23) and matched paracancerous normal tissues (n = 18) in relation to racial/ethnic and age differences. Restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-based sequencing were used to evaluate two regions of mtDNA spanning 5317 to 7608 and 8282 to 10110 bp and including ND subunits 2, 3, MT-COI, II, and III, ATPase 8, a part of ATPase 6, and tRNA genes in frozen ovarian tissues obtained from the southern regional Cooperative Human Tissue Network (CHTN) and University of Alabama-Birmingham (UAB) Ovarian Spore Center. Thirty-nine mtDNA variants were detected of which 28 were previously unreported. One somatic variant of C9500T was observed. A variant, C7028T in the MT-CO1 gene, had an ascending frequency from borderline (8%) to stages III/IV (75%) among the three ovarian cancer subtypes and stages. It was found in 86% (42/49) of African-American and 43% (37/87) of the Caucasian women. A variant, T8548G in the ATPase 6 gene was detected at a frequency of 72% (18/25) in ovarian serous subtype tissues in stages III/IV. Of the African American patients under age 40, 95% (20/21) harbored the T8548G variant; this was in contrast to only 22% (8/35) of Caucasian patients in same age group. Variants C7256T and G7520A had a frequency of 54% (6/11) in endometrioid stage III; no corresponding variants were observed in mucinous subtype stage III. Furthermore, variants C7256T and G7520A were absent in serous ovarian cancer subtype. Interestingly, the C7520T variant in tRNA gene was present in 74% (36/49) of African American and 26% (23/87) of Caucasian patients. Taken together, our results suggest that, with respect to ethnic and age difference, these mtDNA variants may be involved in epithelial ovarian carcinogenesis.

Differential Measurements of Oxidatively Modified Proteins in Colorectal Adenopolyps

Introduction Adenopolyps patients have a three-fold higher risk of colon cancer over the general population, which increases to six-fold if the polyps are multiple and with lower survival among African American population. Currently, 6% of CRC can be ascribed to mutations in particular genes. Moreover, the optimal management of patients with colorectal adenopolyps depends on the accuracy of appropriate staging strategies because patients with similar colorectal adenocarcinoma architecture display heterogeneity in the course and outcome of the disease. Oxidative stress, due to an imbalance between reactive oxygen species (ROS) and antioxidant capacities as well as a disruption of redox signaling, causes a wide range of damage to DNA, proteins, and lipids which promote tumor formation. Objective/Method This study applied spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two-dimensional gel electrophoresis, and western blot analyses to assess the levels of oxidatively modified proteins in 41 pairs of primary colorectal tissues including normal/surrounding, adenopolyps (tubular, tubulovillous, villous, polypvillous) and carcinoma. Analysis of variance (ANOVA) and Student’s t-tests were utilized for the resulting data set. Results Our data showed that the levels of reactive protein carbonyl groups significantly increased as colorectal adenopolyps progresses to malignancy. No significant differences were found in the levels of carbonyl proteins between gender samples analyzed. For African American patients, there were, relative to Caucasians, 10% higher levels of reactive carbonyls in proteins of tubulovillous tissue samples (P < 0.05) and over 36% higher in levels in adenocarcinomas (P < 0.05). In normal tissues and tubular, there were no significant differences between the two groups in levels of protein carbonyls. Differences in the levels of protein carbonyl expression within individual patient samples with different number of tumor cells were notably evident. Conclusion Results suggested that oxidative stress could be involved in the modification of oxidatively carbonyl proteins in the precancer stages, leading to increased aggressiveness of colorectal polyps.

Oxidatively Modified Proteins in the Serous Subtype of Ovarian Carcinoma

Serous subtype of ovarian cancer is considered to originate from fallopian epithelium mucosa that has been exposed to physiological changes resulting from ovulation. Ovulation influences an increased in inflammation of epithelial ovarian cells as results of constant exposure of cells to ROS. The imbalance between ROS and antioxidant capacities, as well as a disruption of redox signaling, causes a wide range of damage to DNA, proteins, and lipids. This study applied spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two-dimensional gel electrophoresis, and Western blot analyses to assess the levels of oxidatively modified proteins in 100 primary serous epithelial ovarian carcinoma and normal/surrounding tissues. These samples were obtained from 56 Caucasian and 44 African-American patients within the age range of 61 ± 10 years. Analyses showed that the levels of reactive protein carbonyl groups increased as stages progressed to malignancy. Additionally, the levels of protein carbonyls in serous ovarian carcinoma among African Americans are 40% (P < 0.05) higher relative to Caucasian at similar advanced stages. Results suggest that oxidative stress is involved in the modification of carbonyl protein groups, leading to increased aggressiveness of epithelial ovarian tumors and may contribute to the diseases invasiveness among African Americans.

Expression of mitochondrial genes MT-ND1, MT-ND6, MT-CYB, MT-COI, MT-ATP6, and 12S/MT-RNR1 in colorectal adenopolyps

Despite improvements in treatment strategies, colorectal cancer (CRC) still has high mortality rates. Most CRCs develop from adenopolyps via the adenoma-carcinoma sequence. A mechanism for inhibition of this sequence in individuals with a high risk of developing CRC is urgently needed. Differential studies of mitochondrial (mt) gene expressions in the progressive stages of CRC with villous architecture are warranted to reveal early risk assessments and new targets for chemoprevention of the disease. In the present study, reverse transcription-quantitative PCR (RT-qPCR) was used to determine the relative amount of the transcripts of six mt genes [MT-RNR1, MT-ND1, MT-COI, MT-ATP6, MT-ND6, and MT-CYB (region 648–15887)] which are involved in the normal metabolism of mitochondria. A total of 42 pairs of tissue samples obtained from colorectal adenopolyps, adenocarcinomas, and their corresponding adjacent normal tissues were examined. Additionally, electron transport chain (ETC), complexes I (NADH: ubiquinone oxidoreductase) and III (CoQH2-cytochrome C reductase), and carbonyl protein group contents were analyzed. Results indicate that there were differential expressions of the six mt genes and elevated carbonyl protein contents among the colorectal adenopolyps compared to their paired adjacent normal tissues (p < 0.05). The levels of complexes I and III were higher in tumor tissues relative to adjacent normal tissues. Noticeably, the expression of MT-COI was overexpressed in late colorectal carcinomas among all studied transcripts. Our data suggest that increased expressions in certain mt genes and elevated levels of ROS may potentially play a critical role in the colorectal tumors evolving from adenopolyps to malignant lesions.

Gonadotropin-Dependent Neuregulin-1 Signaling Regulates Female Rat Ovarian Granulosa Cell Survival

&NA; Mammalian ovarian follicular development and maturation of an oocyte competent to be fertilized and develop into an embryo depends on tightly regulated, spatiotemporally orchestrated crosstalk among cell death, survival, and differentiation signals through extra‐ and intraovarian signals, as well as on a permissive ovarian follicular microenvironment. Neuregulin‐1 (NRG1) is a member of the epidermal growth factor‐like factor family that mediates its effects by binding to a member of the erythroblastoma (ErbB) family. Our experimental results suggest gonadotropins promote differential expression of NRG1 and erbB receptors in granulosa cells (GCs), and NRG1 in theca cells during follicular development, and promote NRG1 secretions in the follicular fluid (FF) of rat ovaries. During the estrous cycle of rat, NRG1 and erbB receptors are differentially expressed in GCs and correlate positively with serum gonadotropins and steroid hormones. Moreover, in vitro experimental studies suggest that the protein kinase C inhibitor staurosporine (STS) causes the physical destruction of GCs by the activation of caspase‐3. Exogenous NRG1 treatment of GCs delayed onset of STS‐induced apoptosis and inhibited cleaved caspase‐3 expressions. Moreover, exogenous NRG1 treatment of GCs alters STS‐induced death by maintaining the expression of ErbB2, ErbB3, pAkt, Bcl2, and BclxL proteins. Taken together, these studies demonstrate that NRG1 is gonadotropin dependent, differentially regulated in GCs and theca cells, and secreted in ovarian FF as an intracellular survival factor that may govern follicular maturation.

Abstract 5168: Mitochondrial 16srRNA variants and complex III expression in serous ovarian cancer

Patients suffering from serous ovarian cancer (SOC) are typically diagnosed at later stage, drastically decreasing their five-year survival prospect. Diagnostic tools, including the measurement of elevated levels of the CA125 protein released by ovarian tumors, provide valuable information but are inherently limited in specificity in diverse populations. Hence, there remains a need for more advanced diagnostic tests to distinguish the different histopathological subclasses to improve treatment outcome. Reactive oxygen species (ROS), by products of normal energy production within the mitochondria, are among the list of compounds believed to be involved in aging and the pathogenesis of many human diseases. Moreover, elevated ROS levels may cause subtle changes in regions of the mitochondrial genome (mtDNA) inducing oxidative stress and tumor microenvironment. Consequently, the events could be used to establish potential mtDNA markers involved in the progression of SOC as an age related disease. The aim of this study is to determine mitochondrial mutational roles and protein expression level in both precancerous and malignant stages of SOC. In the current study PCR-based sequencing was used to detect mtDNA sequence variants in the 16srRNA gene, known to be highly mutated in SOC. The gene spans 1671 to 3229 bp within the mtDNA and is highly conserved. Thirty-one frozen SOC tissue samples of four histopathological subclasses [cystadenoma, n = 7; borderline, n = 8; and malignant stages III/IV, n = 8 and normal no egg, no surface epithelium as a control, n = 8] were examined. Additionally, expression of mitochondrial complex III (Coenzyme Q-cytochrome c reductase) was evaluated using western blot analysis. Forty variants were detected of which three were unreported. G1811A, an unreported variant was detected in 50% of the borderline samples. The unreported variant C2794G was detected in normal, cystadenoma, and borderline with an increasing frequency of 50%, 83% and 100% correspondingly, however did not display in the malignant samples. A3364G an unreported variant was observed in all four categories with a frequency of approximately 70% or higher. Complex III was expressed progressively in normal, borderline and malignant samples. Moreover, the expression of complex III was significantly higher in the malignant samples compared to the normal control. Results from this study could suggest that the genetic instability of 16srRNA region may play a role in ovarian tumorigenicity. Additionally the over expression of complex III in the malignant stages could serve as a target for chemotherapy of serous ovarian disease. Citation Format: Shakeria L. Cohen, Sharifeh Mehrabi, Edward E. Partridge, Felix Aikhionbare. Mitochondrial 16srRNA variants and complex III expression in serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5168.

Abstract 5163: Mitochondrial mutations and gene expression analysis in colorectal adenopolyps

Colorectal cancer (CRC) is the third most common diagnosed and cause of cancer related deaths in both men and women in the United States. Numerous studies have analyzed mitochondria DNA mutations in CRC and other tumors. Results from these studies have detected high mutation rates which may lead to mitochondrial deregulation and tumor progression. Most CRCs develop from adenopolyps via the adenoma-carcinoma sequence. Therefore, analysis of mitochondrial mutations and gene expression may provide a mechanism for inhibition of this tumoral sequence in individuals with a high risk of developing CRC. In the present study, PCR based sequencing and reverse transcription-quantitative PCR (RT-qPCR) were used to determine if mutations in mitochondrial encoded genes and levels of expression of these genes could influence the progression of the adenoma- carcinoma tumoral sequence. Genes analyzed included MT-RNR1, MT-COI, MT-ATP6, MT- MT-CYB, and mitochondrial ND genes that are involved in the normal metabolism of mitochondria. Measurements were made for 34 tissue sample pairs obtained from various types of colorectal adenomas and corresponding normal surrounding tissues. Additionally, mitochondrial complexes I (NADH: ubiquinone oxidoreductase) and III (CoQH2-cytochrome c reductase) protein was analyzed. There was progressive differential expression of mt-genes and complexes I and III proteins among the colorectal tumor stages relative to their paired normal samples. The level of complexes I and III were higher in tumor tissues relative to normal surrounding tissues. Noticeably, the expression of MT-COI was higher in late stage carcinomas among; all studied transcripts. We detected 54 point mutations in one region ranging from 11871-11877. The frequency of these mutations in all stages was as followed; 71.5% in tubular adenoma, 57% tubulovillous, and 43% in villous and carcinoma. Our results suggest that alterations in mt-gene expression play a role in the transformation of the colorectal tumoral stages. Citation Format: LaShanale M. Wallace, Sharifeh Mehrabi, Xuebiao Yao, Felix Aikhionbare. Mitochondrial mutations and gene expression analysis in colorectal adenopolyps. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5163.

Abstract 5262: Specific mtDNA variants and colorectal adenopolyps

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The clinical frequency of colorectal cancer occurrence increase in adenomas with a predominant villous histopathology: tubular (5%), tubulovillous (22%) and villous (40%). Adenoma may develop throughout the gastrointerestinal tract with coarse lobulations formed by branching tubular glands whose lumens open on the surface which are rare in most patient gastrointerestinal tract areas other than the colorectum. The adenoma-carcinoma sequence is clinically associated in patients with polyposis and ulcerative colitis since their predilection for developing carcinoma in adenomatous epithelium is greatly increased. This study focused on COX, ATPase, rRNA, Cytb and ND genes of the mitochondrial genome that have been associated as modifying risks of cancer. Using PCR-based sequence and High resolution restriction endonuclease, we investigated the potential role of mtDNA alterations in colorectal adenopolyps (Tubular, Tubulovillous, and Villous) and carcinoma. Fifty-eight primary tissues of colorectal adenopolyps, carcinoma and their matched normal controls were used in this study. Twenty-two variants were observed and most of these variants were germline and homoplasmic transitions. Unreported germline variants Cytb-A14782T (90%) and MT-ATPase 8 A9169C (75%) were associated with tubular adenomas. Also, MT-ATPase 6- variant T8994A (85%) was associated with tubulovillous adenomas. Moreover, in the highly conservative region of rRNA, variant T1738C was found in colorectal carcinoma only. Results suggest that some specific mtDNA variants may exert their effects on colorectal adenopolyps to gain a proliferative advantage in patients bearing CRC. Citation Format: LaShanale M. Wallace, Sharifeh Mehrabi, Xuebiao Yao, Felix O. Aikhionbare. Specific mtDNA variants and colorectal adenopolyps. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2015-5262