Mohamed M. Desouki

Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion

Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (ρ0 cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and ρ0 epithelial cells. The focal proteins in these networks include i) FN1 (fibronectin) and ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 5 of 6 members of peroxiredoxin whose expression were altered in ρ0 epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect the chromosomal stability. Consistent with above finding our study revealed an increase DNA double strand breaks, and unique chromosomal rearrangements in ρ0 breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that i) claudin-1 and 7 were indeed down regulated in ρ0 breast epithelial cells, ii) down regulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and iii) claudin-1 and -7 were also down regulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells

Proteomic analysis of proteins involved in mitochondria-to-nucleus retrograde response in human cancer cells

All tumors examined to date contain mutations in mitochondrial DNA (mtDNA). In addition, depletion of mtDNA is reported in a variety of tumors. Mitochondrial dysfunction resulting from changes in mtDNA invokes mitochondria-to-nucleus retrograde response in human cells. To identify proteins involved in retrograde response and their potential role in tumorigenesis, we carried out a comparative proteomic analysis using a cell line in which the mitochondrial genome was completely depleted (?0 cells lacking all mtDNA-encoded protein subunits), a cybrid cell line in which mtDNA was restored, and the parental cell line. Our comparative proteomic approach revealed marked changes in the cellular proteome and led us to identify quantitative changes in expression of several proteins. We found that subunits of complex I and complex III, molecular chaperones, and a protein involved in cell cycle control were down-regulated and Inosine 5’-monophosphate dehydrogenase type 2 (IMPDH2) involved in nucleotides biosynthesis was up-regulated in ?0 cells. Our findings demonstrate that the expression of proteins is restored to wild type level by transfer of wild type mitochondria to ?0 cells, suggesting that these proteins play key roles in retrograde response. To determine a potential role for identified retrograde responsive proteins in tumorigenesis, we analyzed the expression of UQCRC1 gene (encoding ubiquinol cytochrome-c reductase core protein I) in breast and ovarian tumors. We found that 1) UQCRC1 was highly expressed in breast (74%) and ovarian tumors (34%) and 2) the expression positively correlated with cytochrome c-oxidase (COXII) encoded by mtDNA. Our study opens an avenue for identification of retrograde proteins as potential tumor suppressors or oncogenes involved in carcinogenesis.

Frequent expression of napsin a in clear cell carcinoma of the endometrium: Potential diagnostic utility

The histotyping of high-grade endometrial carcinomas with clear cells may be subject to significant interobserver variability, which suggests that a biomarker that can distinguish endometrial clear cell carcinoma (CCC) from its mimics would be of diagnostic utility. This study assessed the usefulness of napsin A immunohistochemistry in the diagnosis of CCC, on the basis of an analysis of 77 cases diagnosed as such at 9 institutions. After being independently reviewed by a subset of 3 pathologists, cases for which there was diagnostic consensus among all 3 reviewers in agreement with the primary contributor (n=60) were used to establish a “consensus group” that served as a gold standard relative to which napsin A performance was assessed. Duplicate, 1.0-mm-core tissue microarrays were constructed from the 54 cases in the consensus group for which requisite materials were available, as well as from 49 endometrial endometrioid carcinomas (all grades) and 17 endometrial serous carcinomas. Napsin A immunohistochemical analysis was performed on the microarrays and on the 17 cases for which there was no diagnostic consensus, with scoring based on the proportion of immunoreactive cells (0, 1+, 2+, and 3+ indicative of 0, 1% to 25%, 26% to 49%, and ≥50% immunoreactive cells, respectively). The distribution of scores for the 49 CCC cases with evaluable cores was as follows: 0, n=6; 1+, n=6; 2+, n=8; 3+, n=29. Among the evaluable cases, the frequency of ≥1+ napsin A immunoreactivity was significantly higher in CCCs (43/49, 88%) than in endometrial serous carcinomas (1/13, 7.7%; P<0.0001) and endometrial endometrioid carcinomas (0/49, 0%; P<0.0001). The sensitivity, specificity, negative predictive value, and positive predictive value of ≥1+ napsin A expression in predicting the consensus clear cell histotype were 0.88 (95% confidence interval [CI], 0.75-0.95), 0.98 (95% CI, 0.9-1), 0.91 (95% CI, 0.86-0.96), and 0.98 (95% CI, 0.86-1), respectively. Napsin A expression was not associated with survival or clinicopathologic factors. In the group of cases without diagnostic consensus for CCC, 50% showed ≥1+ napsin A expression; all napsin A-negative cases had previously been classified as non-CCC by ≥2 reviewers, whereas only 37.5% of the napsin A-positive cases had been classified as CCC by 2 of the 3 reviewers. In conclusion, napsin A is a sensitive and specific biomarker of the clear cell histotype in endometrial carcinomas and accordingly may have diagnostic utility in their histotyping.

The clinicopathologic significance of p53 and BAF-250a (ARID1A) expression in clear cell carcinoma of the endometrium.

TP53 mutation (and associated p53 protein overexpression) is probably a negative prognostic marker in endometrial cancers, but its relevance in the rarer histologic subtypes, including clear cell carcinomas, has not been delineated. Preclinical studies suggest functional interactions between p53 and the BAF250a protein, the product of a tumor suppressor gene ARID1A (adenine-thymine (AT)-rich interactive domain containing protein 1A) that is frequently mutated in ovarian clear cell carcinoma. In this study, we evaluated the significance of p53 and BAF250a expression, as assessed by immunohistochemistry, in a group of 50 endometrial clear cell carcinomas. Of 50 cases, 17 (34%) were p53+, and the remaining 33 cases had a p53 wild-type (p53-wt) immunophenotype. Of the 11 relapses/recurrences in the entire data set, 73% were in the p53+ group (P=0.008). On univariate analyses, the median overall survival for the p53-wt patients (83 months) was longer than the p53+ patients (63 months) (P=0.07), and the median progression-free survival for the p53-wt group (88 months) was significantly longer than the p53+ group (56 months) (P=0.01). On multivariate analyses, p53 expression was not associated with reduced overall or progression-free survival. In addition, p53 status was not significantly associated with pathologic stage or morphologic patterns. Of the 50 cases, 10 (20%) showed a complete loss of BAF250a expression. There was no significant correlation between p53 and BAF250a expression. The p53+/BAF250a−, p53+/BAF250a+, p53-wt/BAF250a+ and p53-wt/BAF250a− composite immunophenotypes were identified in 8%, 26%, 54% and 12% of cases, respectively, and neither loss of BAF250a expression nor composite p53/BAF250a expression patterns were associated with reduced overall or progression-free survival. In conclusion, a significant subset of CCC express p53, and these cases are apparently not definable by their morphologic features. P53 expression may be a negative prognostic factor in this histotype, and warrants additional studies. Loss of BAF250a expression has no prognostic significance in endometrial clear cell carcinomas.

Comparative analysis of Napsin A, alpha-methylacyl-coenzyme A racemase (AMACR, P504S), and hepatocyte nuclear factor 1 beta as diagnostic markers of ovarian clear cell carcinoma: an immunohistochemical study of 279 ovarian tumours

Summary Napsin A and &agr;-methylacyl-coenzyme A racemase (AMACR, P504S) have recently been described as being frequently expressed in clear cell carcinomas (CCC) of the gynecological tract. The present study was conducted to assess the test performance of these newer markers relative to the more traditional marker, hepatocyte nuclear factor 1&bgr; (HNF1&bgr;), in a large and histotypically diverse dataset. A total of 279 ovarian tumours in tissue microarrays were immunohistochemically assessed for the expression of Napsin A, AMACR and HNF1&bgr;. HNF1&bgr;, Napsin A and AMACR were expressed in 92%, 82% and 63% of 65 CCC, 7%, 1% and 1% of 101 serous carcinomas, 37%, 5.3% and 0% of 19 endometrioid carcinomas, 60%, 0% and 0% of 45 mucinous tumours, 100%, 0% and 0% of seven yolk sac tumours, and 0%, 16.7% and 16.7% of six steroid cell tumours NOS, respectively. All other tumours, including 18 adult-type granulosa cell tumours, eight dysgerminomas and nine other miscellaneous tumour types were negative for all three markers. Using a benchmark of ≥1% of tumour cells for positivity and CCC as the diagnostic end-point, the sensitivity, specificity, negative predictive value and positive predictive value of Napsin A expression were 0.82, 0.99, 0.94, and 0.98, respectively (odds ratio 439, p < 0.0001). Respective parameters were 0.92, 0.79, 0.97, and 0.58 (odds ratio 44, p < 0.0001) for HNF1&bgr; and 0.63, 0.99, 0.89, and 0.5 (odds ratio 112, p < 0.0001) for AMACR. The combination of any two positive markers, irrespective of the staining pattern of the third, significantly predicted the CCC histotype in every analytic scenario. In summary, HNF1&bgr; is highly sensitive but is suboptimally specific in isolation, whereas AMACR is highly specific but is suboptimally sensitive. Napsin A is specific but of intermediate sensitivity. Napsin A, AMACR and HNF1&bgr; are all viable markers of CCC that can be deployed as components of larger panels when CCC is a diagnostic consideration.

Incidental atypical proliferative lesions in reduction mammoplasty specimens: analysis of 2498 cases from 2 tertiary women's health centers ☆

Atypical proliferative lesions (APLs) are occasionally found in breast reduction specimens. The aim of the study was to investigate the prevalence of APL in reduction mammoplasty specimens from patients who were treated mainly for macromastia. A retrospective medical record review of pathology records on patients who underwent reduction mammoplasty from 2006 to 2012 generated 2498 cases. The sole exclusion criterion was a history of invasive and/or ductal carcinoma in situ (DCIS). Laterality, specimen weight, number of blocks submitted, and presence of APL were recorded and analyzed. We defined APL as invasive carcinoma, DCIS or lobular carcinoma in situ, atypical ductal (ADH) or lobular hyperplasia, and flat epithelial atypia (FEA). The presence of papillomas, radial scars, and fibroadenomas was also recorded. At least 1 APL was identified in 107 (4.3%) of 2498 reduction mammoplasty specimens including invasive duct carcinoma (n = 2), DCIS (n = 4), ADH/FEA (n = 47), and lobular carcinoma in situ/atypical lobular hyperplasia (n = 54). One hundred four (97%) of the 107 patients underwent bilateral, and 3 (3%) underwent unilateral reductions. In conclusion, the frequency of detection of APLs in patients with no history of breast cancer is low (4.3%). Detection of invasive and DCIS lesions is extraordinarily low at 0.2%. The most common APL is lobular neoplasia (2.2%), whereas ADH and FEA are seen in 1.9%. Our findings provide data on the distribution of these lesions in this setting, as well as some insight into their prevalence in the general population. A protocol for submitting tissues from these specimens is also proposed.

Src Kinase and Mitogen-Activated Protein Kinases in the Progression from Normal to Malignant Endometrium

Purpose: The purpose of this research was to determine whether a correlation exists between the levels of activated mitogen-activated protein kinase (MAPK) and Src kinases and the progression from normal to malignant endometrium. Experimental Design: We measured total and phosphorylated levels for extracellular signal-regulated kinase 1/2, p38, stress-activated protein kinase/c-Jun NH2-terminal kinase, and Src kinases from 33 frozen endometrial adenocarcinomas and 38 benign endometrial specimens by quantitation of signals from Western blots using antibodies against these kinases. Results: Elevated phospho-extracellular signal-regulated kinase 1/2 (150 ± 40 versus 46 ± 7; P = 0.03), phospho-Src (28 ± 5 versus 4 ± 1), and phospho-p38 (131 ± 16 versus 27 ± 7; P < 0.001) was detected in benign versus malignant endometrium when the Western blot signal of activated kinase was normalized to total kinase levels and β actin. A modest increase in active c-Jun NH2-terminal kinase was detected in carcinoma versus benign specimens (51 ± 13 versus 43 ± 10; P = 0.8). Expression of total kinases (normalized to β-actin) was higher in carcinoma versus benign specimens, respectively (extracellular signal-regulated kinase 1/2, 9 ± 2 versus 0.7 ± 0.1; Src, 7 ± 2 versus 0.4 ± 0.1; stress-activated protein kinase c-Jun NH2-terminal kinase, 2 ± 0.4 versus 0.2 ± 0.02; P < 0.001; and p38, 1 ± 0.2 versus 0.4 ± 0.1; P < 0.01). Immunohistochemistry for active and total Src kinases and MAPKs detected positive staining in epithelial and stroma cells. Conclusions: These data demonstrated that, in contrast with breast cancer, the progression from normal to malignant endometrium is not associated with activation of MAPK and Src kinases. Elevation of these active kinases in benign endometrium may contribute to endometrial resistance to the antiestrogen action of tamoxifen.

Original contributionDecreased mitochondrial SIRT3 expression is a potential molecular biomarker associated with poor outcome in breast cancer

SIRT3 is a genomically expressed, mitochondrial localized tumor suppressor protein where it directs multiple metabolic processes by deacetylating downstream protein substrates. Genetic deletion of Sirt3 in mice leads to the spontaneous development of mammary tumors starting at 1 year, and decreased SIRT3 messenger RNA has been observed in several human tumors including breast malignancies. In this investigation, we assessed SIRT3 expression in human breast cancer tissue microarray and examined the relationship between SIRT3 expression and outcome in patients with breast cancer. SIRT3 protein expression is significantly lower in neoplastic compared with normal breast epithelium, including normal epithelium adjacent to tumors. Patients with breast cancer in the lowest quartile of SIRT3 expression had a significantly shorter locoregional relapse-free survival (hazard ratio, 0.53 [0.47-0.61]; log-rank P = 0). Notably, low SIRT3 expression was associated with reduced locoregional relapse-free survival in all breast cancer subtypes analyzed, including ER+, ER-, HER2+, and basal subtypes (hazard ratios, 0.44-0.65; log-rank P = 0-.0019). These results highlight the importance of the SIRT3 as a tumor suppressor protein in breast cancer and suggest that SIRT3 may be a potential molecular biomarker to identify high-risk patients across all molecular subtypes of breast cancer.

Expression of the oncofetal protein IGF2BP3 in endometrial clear cell carcinoma: assessment of frequency and significance

Insulin-like growth factor-II messenger RNA-binding protein 3 (IGF2BP3 or IMP3) is a biomarker whose expression has been found to be a negative prognostic factor in several neoplasms including ovarian clear cell carcinoma (CCC). In this study, we analyzed the frequency and clinicopathologic significance of IMP3 expression, as assessed by immunohistochemistry and as scored using a modified H-score system, in a cohort of 50 endometrial CCCs. Cases with scores of 0 to 100, 101 to 200, and 201 to 300 were classified as negative/mildly positive (n = 17), moderately positive (n = 20), and strongly positive (n = 13), respectively. A distinctive pattern of increased staining at the myoinvasive front (relative to the main tumor) was evident in 46% of the cases with evaluable foci of myometrial invasion. Moderate/strong IMP3 staining was associated with a tumor architectural pattern that has been reported to be of poor prognostic significance: at least 10% of the tumor composed of solid architecture or individual infiltrating tumor cells (P = .01). Increasing levels of IMP3 expression showed a trend toward decreasing relapse-free survival (RFS; median survival, 75.6, 81.3, and 48.4 months for the negative/mildly, moderately, and strongly positive groups, respectively [P = .09]). However, IMP3 expression was not significantly associated with reduced overall survival or RFS in a multivariate analytic model. The finding in a subset of our cases of increased IMP3 expression at the tumoral myoinvasive front is consistent with a role for IMP3 in invasiveness, as is the trend toward reduced RFS in cases expressing IMP3 at high levels. These preliminary findings suggest that IMP3 expression may be involved in the pathogenesis of CCC and is worthy of further exploration.

Utility of α-methylacyl-coenzyme-A racemase (p504s) immunohistochemistry in distinguishing endometrial clear cell carcinomas from serous and endometrioid carcinomas

The expression of α-methylacyl-coenzyme-A racemase (AMACR) has previously been reported in 75% to 100% of urethral/bladder clear cell carcinomas, tumors that are known to display broad phenotypic overlap with their identically named müllerian counterparts. Herein, we assess the utility of AMACR in distinguishing endometrial clear cell carcinomas (CCCs) from endometrial serous carcinomas (ESCs) and endometrial endometrioid carcinomas (EECs). A total of 111 endometrial carcinomas in a tissue microarray, including 49 CCCs, 13 ESCs, and 49 EECs, were assessed for AMACR immunoreactivity, with results scored semiquantitatively (scores 0, 1+, 2+, 3+ for 0%, 1%-5%, 6%-50%, >50% immunoreactive cells, respectively). Fifty (45%) of the 111 carcinomas were AMACR positive, with the following score distribution: CCC: 0 (n = 12), 1+ (n = 12), 2+ (n = 3), 3+ (n = 22); EEC: 0 (n = 38), 1+ (n = 4), 2+ (n = 4), 3+ (n = 3); ESC: 0 (n = 11), 1+ (n = 1), 2+ (n = 0), 3+ (n = 1). AMACR expression was significantly more frequent in CCC (75%) than in ESC (15%) or EEC (22%); P < .0001. The sensitivity and specificity of AMACR expression in classifying a carcinoma as CCC were 0.75 (95% confidence interval [CI], 0.61-0.86) and 0.79 (95% CI, 0.66-0.88), respectively, with an odds ratio of 11.62 (95% CI, 5-28; P < .001) and an area under the curve of 0.79 (95% CI, 0.68-0.88). These findings indicate that AMACR expression is strongly associated with CCC and displays a relatively robust diagnostic test performance. However, its practical utility may be limited by the focal nature of its expression in 32% of the AMACR-positive CCC cases as well as its expression in 15% to 22% of the non-CCC histotypes.