Annu Makker

PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update

Abnormalities in ovarian function, including defective oogenesis and folliculogenesis, represent a key female reproductive deficiency. Accumulating evidence in the literature has shown that the PI3K/PTEN/Akt and TSC/mTOR signaling pathways are critical regulators of ovarian function including quiescence, activation, and survival of primordial follicles, granulosa cell proliferation and differentiation, and meiotic maturation of oocytes. Dysregulation of these signaling pathways may contribute to infertility caused by impaired follicular development, intrafollicular oocyte development, and ovulation. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/Akt and TSC/mTOR pathways during mammalian oogenesis and folliculogenesis and their association with female infertility.

VEGF-A immunohistochemical and mRNA expression in tissues and its serum levels in potentially malignant oral lesions and oral squamous cell carcinomas

The aim of the study was to investigate whether the estimation of circulating Vascular endothelial growth factor-A (VEGF-A) levels by ELISA could be used as surrogate of VEGF-A expression in tissues of pre-malignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC) as compared to that in healthy controls. The study samples comprised of tissue and blood samples from 60 PMOLs, 60 OSCC, and 20 healthy controls. Serum VEGF-A levels were determined by an ELISA based assay (Quantikine human VEGF; R & D System, Minneapolis USA). Tissue VEGF-A expression and microvessel density (MVD) were assessed by immunohistochemistry (IHC) using antibodies against VEGF-A and CD-34 on formalin fixed paraffin embedded (FFPE) tissue sections. VEGF-A mRNA expression was analyzed by real-time PCR in snap frozen tissues. Serum VEGF-A levels and immunohistochemical VEGF-A expression were significantly high in PMOLs and OSCC in comparison with controls. VEGF mRNA gene expression showed more than 50-fold increase in PMOLs and OSCC. VEGF-A levels in serum correlated in a linear fashion with the tissue expression in oral pre-malignant and malignant lesions, suggesting that the serum levels may serve as surrogate material for tissue expression of VEGF-A.

Tumor progression, metastasis, and modulators of epithelial–mesenchymal transition in endometrioid endometrial carcinoma: an update

Endometrioid endometrial carcinoma (EEC), also known as type 1 endometrial cancer (EC), accounts for over 70-80% of all cases that are usually associated with estrogen stimulation and often develops in a background of atypical endometrial hyperplasia. The increased incidence of EC is mainly confined to this type of cancer. Most EEC patients present at an early stage and generally have a favorable prognosis; however, up to 30% of EEC present as high risk tumors, which have invaded deep into the myometrium at diagnosis and progressively lead to local or extra pelvic metastasis. The poor survival of advanced EC is related to the lack of effective therapies, which can be attributed to poor understanding of the molecular mechanisms underlying the progression of disease toward invasion and metastasis. Multiple lines of evidence illustrate that epithelial-mesenchymal transition (EMT)-like events are central to tumor progression and malignant transformation, endowing the incipient cancer cell with invasive and metastatic properties. The aim of this review is to summarize the current knowledge on molecular events associated with EMT in progression, invasion, and metastasis of EEC. Further, the role of epigenetic modifications and microRNA regulation, tumor microenvironment, and microcystic elongated and fragmented glands like invasion pattern have been discussed. We believe this article may perhaps stimulate further research in this field that may aid in identifying high risk patients within this clinically challenging patient group and also lead to the recognition of novel targets for the prevention of metastasis - the most fatal consequence of endometrial carcinogenesis.

Presence of CD3+ tumor infiltrating lymphocytes is significantly associated with good prognosis in infiltrating ductal carcinoma of breast.

BACKGROUND Aim of this study was to investigate the prognostic significance of CD3+ TILs in infiltrating ductal carcinoma (IDC) of the breast. MATERIALS AND METHODS Immuno-histochemistry was done with CD3 antibodies in tissue sections of 127 breast cancer patients, and CD3+ intra-tumoral and stromal TILs were counted in relation to clinico-pathological variables. RESULTS Intra-tumoral and stromal CD3+ TILs were significantly associated with positive lymph node status (P = 0.006, P = 0.043, respectively) without significant association with age, menopausal status, family history, and hormonal status. The higher CD3 intra-tumoral and stromal counts both showed significant association with good prognosis (P = 0.039, P = 0.044, respectively). The intra-tumoral count was higher than stromal count and was independently associated with disease-free survival in stage I and II cancer (P = 0.021). CONCLUSIONS CD3+ TILs may serve as independent marker of good prognosis in IDC breast. The findings of this study need further validation on a larger sample size.

Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

Endometrial Expression of Homeobox Genes and Cell Adhesion Molecules in Infertile Women With Intramural Fibroids During Window of Implantation.

This study was designed to examine the expression and cellular distribution of homeobox (HOX) genes (HOXA10 and HOXA11) and cell adhesion molecules (E-cadherin, N-cadherin, and β-catenin) during the window of implantation in infertile women with noncavity-distorting intramural (IM) fibroids (n = 18) and in fertile controls (n = 12). Quantitative real-time polymerase chain reaction and immunohistochemistry were used to evaluate the messenger RNA (mRNA) levels and protein expression, respectively. When compared to fertile controls, reduced HOXA10 and HOXA11 transcript and protein levels were observed in infertile women. However, changes only in the expression of HOXA10 mRNA (−1.72-fold; P = .03) and stromal protein (P = .001) were statistically significant. Significantly lower E-cadherin mRNA (−10.97-fold; P = .02) and protein levels were seen in infertile patients. E-cadherin immunostaining was significantly reduced both in the luminal (P = .048) and in the glandular (P = .014) epithelium of endometrium from infertile patients when compared to controls. No significant change was observed either in the mRNA levels or in the immunoexpression of N-cadherin and β-catenin. However, a trend toward lower N-cadherin expression in the luminal epithelium (P = .054) and decreased β-catenin expression in the glandular epithelium (P = .070) was observed in infertile patients. The present findings suggest that altered endometrial HOXA10 and E-cadherin mRNA and protein expression observed in infertile women with IM fibroids during the mid-secretory phase might impair endometrial receptivity leading to infertility in these patients.

Fibroblast Growth Factor (FGF-2) and Its Receptors FGFR-2 and FGFR-3 May Be Putative Biomarkers of Malignant Transformation of Potentially Malignant Oral Lesions into Oral Squamous Cell Carcinoma.

There are several factors like angiogenesis, lymphangiogenesis, genetic alterations, mutational factors that are involved in malignant transformation of potentially malignant oral lesions (PMOLs) to oral squamous cell carcinoma (OSCC). Fibroblast growth factor-2 (FGF-2) is one of the prototypes of the large family of growth factors that bind heparin. FGF-2 induces angiogenesis and its receptors may play a role in synthesis of collagen. FGFs are involved in transmission of signals between the epithelium and connective tissue, and influence growth and differentiation of a wide variety of tissue including epithelia. The present study was undertaken to analyze expression of FGF-2 and its receptors FGFR-2 and FGFR-3 in 72 PMOLs, 108 OSCC and 52 healthy controls, and their role in risk assessment for malignant transformation of Leukoplakia (LKP) and Oral submucous fibrosis (OSMF) to OSCC. Immunohistochemistry was performed using antibodies against FGF-2, FGFR-2 and FGFR-3. IHC results were validated by Real Time PCR. Expression of FGF-2, FGFR-2 and FGFR-3 was upregulated from PMOLs to OSCC. While 90% (9/10) of PMOLs which showed malignant transformation (transformed) expressed FGF-2, only 24.19% cases (15/62) of PMOLs which were not transformed (untransformed) to OSCC expressed FGF-2. Similarly, FGFR-2 expression was seen in 16/62 (25.81%) of untransformed PMOLs and 8/10 (80%) cases of transformed PMOLs. FGFR-3 expression was observed in 23/62 (37.10%) cases of untransformed PMOLs and 6/10 (60%) cases of transformed PMOLs. A significant association of FGF-2 and FGFR-2 expression with malignant transformation from PMOLs to OSCC was observed both at phenotypic and molecular level. The results suggest that FGF-2 and FGFR-2 may be useful as biomarkers of malignant transformation in patients with OSMF and LKP.

Uterine Leiomyomas Effects on Architectural, Cellular, and Molecular Determinants of Endometrial Receptivity

Impaired endometrial receptivity is an important contributing factor to implantation failure. Uterine leiomyomas are widely prevalent steroid hormone–dependent benign tumors that act as a restraint to conception and successful outcome of pregnancies. Reports are available, which suggest that leiomyomas have negative influence on endometrial receptivity to blastocyst implantation. The aim of the present review is to provide a comprehensive picture of the current knowledge of the effect of uterine leiomyomas on the architectural, cellular, and molecular determinants of endometrial receptivity. Understanding the potential role of these factors will provide insight into the underlying mechanisms of leiomyoma-associated infertility and provide new areas for basic and translational research.

Is the Tumor Infiltrating Natural Killer Cell (NK-TILs) Count in Infiltrating Ductal Carcinoma of Breast Prognostically Significant?

PURPOSE The aim of this study was to investigate the prognostic significance of the CD56+NK-TIL count in infiltrating ductal carcinoma (IDC) of breast. MATERIAL AND METHODS Immunohistochemistry (IHC) was performed using antibodies specific for CD56 on formalin-fixed and paraffin-embedded tissue sections of 175 infiltrating ductal carcinomas (IDC) of breast. Distribution of intratumoral and stromal CD56+NK-TILs was assessed semi-quantitatively. RESULTS A low intratumoral CD56+count showed significant and inverse associations with tumor grade, stage, and lymph node status, whereas it had significant and direct association with response to treatment indicating good prognosis. These patients had better survival (χ2=4.80, p<0.05) and 0.52 fold lower death rate (HR=0.52, 95% CI=0.28-0.93) as compared to patients with high CD56+ intratumoral count. The association of survival was insignificant with low CD56 stromal count as compared to high CD56 stromal count (χ2=1.60, p>0.05). CONCLUSION To conclude, although NK-TIL count appeared as a significant predictor of prognosis, it alone may not be sufficient for predicting the outcome considering the fact that there exists a crosstalk between NK-TILs and the other immune infiltrating TILs.

PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas.

Background & objectives: Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. Methods: Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. Results: Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium. Interpretation & conclusions: Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.