Andrzej Semczuk

P16 alterations increase the metastatic potential of endometrial carcinoma.

OBJECTIVE The aim of this study was to investigate the role of p16 in tumorigenesis of endometrial carcinoma (EC). METHODS Expression of p16 protein was analyzed using immunohistochemistry. The methylation status of p16 promoter region was determined by methylation-specific PCR. Deletion analysis of the p16 gene was performed by PCR-analyses. RESULTS Aberrant protein expression of p16 was observed in 18 of 46 (39.2%) ECs and correlated significantly with p16 alterations, including gene deletions in 26 of 46 (56.5%) ECs and promoter hypermethylation in 8 of 46 (17.4%) ECs (p<0.001). A significant increase in the frequency of p16 alterations from early stage (I-II) to advanced stage (III-IV) ECs was observed (p=0.002). There was no significant correlation between p16 protein expression and the clinico-pathological features of EC. The development of metastases correlated significantly with the frequency of p16 alterations: p16 alterations were detected in 14 of 15 (93.3%) PTs with metastases and in only 18 of 31 (58.1%) PTs without metastases (p=0.018). The genetic comparison of 15 primary ECs and their paired metastases revealed that in most of the cases the deleted region of p16 gene remains the same or becomes larger during the progression from primary tumor to its corresponding metastases. CONCLUSION Our results suggest that p16 alterations and particularly p16 gene deletions in ECs are associated with increased incidence of metastases.

Role of GPR30 in endometrial pathology after tamoxifen for breast cancer.

OBJECTIVE This study was undertaken to evaluate the potential role of G-protein-coupled estrogen receptor in endometrial pathology associated with tamoxifen treatment of breast cancer patients. STUDY DESIGN We investigated whether G-protein-coupled estrogen receptor plays a role in mediating proliferating effect of tamoxifen in endometrial carcinoma cells. These results were compared with the G-protein-coupled estrogen receptor expression pattern in endometrial tissue from a cohort of 95 breast cancer patients, who received tamoxifen or another adjuvant therapy. RESULTS In vitro tamoxifen significantly stimulated the mitogen-activated protein kinase phosphorylation and cell proliferation of endometrial cell lines via G-protein-coupled estrogen receptor. In vivo, there was a significant correlation between G-protein-coupled estrogen receptor expression and the tamoxifen-induced endometrial pathology (P = .006). Moreover, G-protein-coupled estrogen receptor positivity was predictive of an earlier development of symptoms, such as bleeding or suspect endometrial thickness, induced by tamoxifen therapy (P = .019). CONCLUSION G-protein-coupled estrogen receptor plays an important role in tamoxifen-induced endometrial abnormalities.

Homoplasmic MELAS A3243G mtDNA mutation in a colon cancer sample

We have analyzed mtDNA variation in various cancer samples, comparing them with normal tissue controls, and identified mutations and polymorphisms, both known and novel, in mitochondrial tRNA, rRNA and protein genes. Most remarkably, in a colon cancer sample we have found the A3243G mutation in the homoplasmic state. This mutation is known to cause severe mitochondrial dysfunction and, until now, has not been found in cancer cells, nor in the homoplasmic state in living subjects. The mutation was absent from normal tissue, suggesting that mtDNA mutation and resulting respiratory deficiency played a role in carcinogenesis.

APC promoter hypermethylation is an early event in endometrial tumorigenesis

The aim of the current study was to investigate the role of promoter methylation of adenomatous polyposis coli (APC) and epithelial cadherin (E‐cadherin) genes in endometrial tumorigenesis. The methylation status of both genes was investigated in 43 cases of normal endometrium, 21 simple hyperplasia, 17 atypical hyperplasia, and 86 endometrial carcinoma (EC). Additionally, the methylation pattern of both genes was analyzed in 24 primary ECs and their corresponding metastases. DNA methylation of the APC gene increased from atypical hyperplasia (23.5%) to endometrial carcinoma, reaching its highest level of 77.4% in early stage cancer (FIGO I and II) and decreasing stepwise to 24.2% in advanced stage carcinomas (FIGO III and IV). No methylation of APC was found in normal endometrium or simple hyperplasia. Methylation of E‐cadherin was found only in EC (22.1%). The mean age of the patients with aberrant APC methylation was 68.8 years and was significantly higher compared to the mean age (60.9 years) of the patients without methylation of APC promoter (P = 0.02). APC promoter methylation significantly correlated with decreased protein expression of APC (P = 0.039), with increased expression of the Ki‐67 proliferative marker (P = 0.006) and decreased metastatic potential (P = 0.002). There was no correlation between APC and E‐cadherin methylation patterns and the other clinicopathologic features, nor with patient outcome. Our results suggest that hypermethylation of APC promoter region is an early event in endometrial tumorigenesis. (Cancer Sci 2009)

GPER-1 Expression Decreases During Breast Cancer Tumorigenesis

GPER-1 protein expression was immunohistochemically examined in 164 primary breast cancer specimens and their matched normal breast epithelium. GPER-1 down-regulation correlated significantly with increased histological grading (p = .015), lymph node metastases (p = .032), and negative estrogen receptor status (p = .018). The decrease of GPER-1 expression in breast cancer tissue, relative to normal tissue, was associated with poor overall survival (p = .043) and disease-free survival (p = .037) and remained a significant unfavorable factor in multivariate analysis for DFS (HR = 1.569; 95% CI, 1.024–2.797; p = .041) and OS (HR = 2.082; 95% CI, 1.248–4.773; p = .039). Thus aberrant GPER-1 expression seems to be an important factor in breast cancer progression.

Expression of TGF-β type I and II receptors in normal and cancerous human endometrium

Transforming growth factor-beta (TGF-beta) belongs to a superfamily of structurally related polypeptides involved in various biological processes, including cell growth, proliferation and differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling. We tried to define the different expression patterns of the TGF-beta receptors by investigating the female reproductive organs during the menstrual cycle and endometrial tumorigenesis, because their role in these processes is still unclear. In this study, we examined the expression of the TGF-beta type I and type II receptors in normal (n=13) and carcinomatous (n=42) endometrial tissue specimens using reverse transcriptase polymerase chain reaction and immunological (Western blot and enzyme linked immunosorbent assay) methods. Two uncommon female genital tract tumors, rhabdomyosarcoma of the uterine cervix and uterine carcinosarcoma, were also included. There were no significant differences between normal and cancerous endometrial tissues regarding the TGF-beta receptors mRNA levels. However, we observed a markedly low TGF-beta type I receptor protein level (P<0.028; Mann-Whitney-U test), while the malignant endometrium showed a significantly higher TGF-beta type II receptor protein level (P<0.007; Mann-Whitney-U test) than the normal endometrium. Moreover, significantly elevated TGF-beta receptor type II protein level was noted when depth of myometrial invasion of endometrial carcinomas was considered (P<0.05; Mann-Whitney-U test). In contrast to uterine carcinosarcoma, in which no detectable mRNA for TGF-beta type II receptor was found, we noted expression of both TGF-beta receptors in rhabdomyosarcoma of the uterine cervix. However, neither rhabdomyosarcoma of the uterine cervix nor uterine carcinosarcoma displayed TGFbetaRI and TGFbetaRII protein expression. This observation corroborates the complexity of the deregulation of TGF-beta receptor expression in human endometrial cancer.

Loss of heterozygosity of the retinoblastoma gene is correlated with the altered pRb expression in human endometrial cancer

Abstract. The retinoblastoma (Rb) gene was the first tumor suppressor gene to be discovered; however, data on the influence of Rb inactivation on endometrial carcinogenesis are scarce. We investigated 46 paired primary human endometrial carcinomas and normal tissues to assess the frequency of loss of heterozygosity (LOH) in Rb and 20 tumor pairs to detect the frequency of p53 LOH. Moreover, expression of the retinoblastoma protein (pRb) was assessed immunohistochemically. Of 44 informative cases 8 showed loss of one allele in at least one Rb marker; Rb LOH frequency thus reached 18%. Two omental metastases of endometrial origin showed a heterogeneity pattern similar to that of the primary tumors. We did not find a significant correlation between Rb LOH and patient age, clinical stage, histological grade or muscle invasion of the tumor. Nevertheless, Rb LOH was demonstrated at early (stage I, 5/27, 18%) and advanced (stages II–IV; 3/9, 33%) clinical stages of the neoplasm, suggesting that LOH at the Rb locus occurs before the clonal expansion of the tumor. There was a significant correlation between Rb LOH and weak/absent pRb expression. We noted a single case of p53 LOH at intron 1, but no tumor showed both alterations simultaneously. Our data suggest that LOH at the Rb locus plays a role in the oncogenesis of a subset of uterine neoplasms and corresponds with the altered expression of the pRb.

Liver X receptor (LXR) and the reproductive system--a potential novel target for therapeutic intervention.

Liver X receptor (LXR) alpha and beta are ligand-activated transcription factors that regulate the expression of genes involved in the removal of cholesterol from cells by high-density lipoproteins, the transport of cholesterol to the liver and the biliary excretion of cholesterol. LXRs are activated by oxygenated cholesterol derivatives such as 24(S),25-epoxycholesterol or 24(S)-, 25- and 27-hydroxycholesterol. In this review, we will discuss the role of LXR in the reproductive system and perspectives on the application of LXR agonists in the treatment of reproductive pathologies. Interestingly, progressive age-related impairment of fertility is observed in both female and male LXR knockout mice. Reduced fertility in female LXR knockout mice is associated with resistance to follicular fluid meiosis-activating sterol (FF-MAS), the intermediate of cholesterol synthesis generated in the ovaries that is responsible for stimulating oocyte meiosis partially in a LXR-dependent manner. Female mice lacking both LXR isoforms exhibit symptoms of ovarian hyperstimulation syndrome when treated with pharmacological doses of gonadotropins. LXR agonists have mainly been considered as potential anti-atherosclerotic medications. However, experimental studies suggest that natural or synthetic LXR agonists may also effectively treat some reproductive pathologies, such as infertility, impaired uterine contractility, hormone-dependent cancers and insulin resistance in patients with polycystic ovarian syndrome. However, the specific adverse effects of LXR agonists on the reproductive system must also be considered. Adverse effects of LXR agonists include impaired trophoblast invasion, excessive transplacental cholesterol transport from the mother to the fetus leading to fetal hypercholesterolemia, and augmented estrogen deficiency after menopause.

Immunohistochemical Analysis of MIB-1 Proliferative Activity in Human Endometrial Cancer. Correlation with Clinicopathological Parameters, Patient Outcome, Retinoblastoma Immunoreactivity and K-Ras Codon 12 Point Mutations

To test the prognostic utility of MIB-1 in human endometrial neoplasias, the proliferative activities of fifty-two endometrial carcinomas obtained from Polish women were assessed. We also investigated the relationship between the MIB-1 Proliferative Index and the well-known clinicopathological features of cancer (clinical stage, histological type, histological grade, depth of myometrial invasion), patients age, overall survival, retinoblastoma immunostaining and K-ras codon 12 point mutations. The mean MIB-1 Proliferation Index was 43.8%, with a median of 36.0%. Due to the great intratumour heterogeneity of the immunoreaction, the Index ranged from 0% to 98%. A significant relationship was noted between MIB-1 expression and histological grading (p=0.0004) and myometrial invasion of cancer (p=0.01). Multivariate Cox regression demonstrated that the clinical stage was the only independent prognostic factor during follow-up (p=0.025). There was a tendency towards a poorer outcome for women with a Proliferative Index of >31% than for patients whose Index was ≤ 31%; the difference, however, did not reach significance (p=0.25; log-rank test). Interestingly, uterine cancers lacking retinoblastoma protein expression had a mean MIB-1 Proliferation Index that was nearly twice as high as in those neoplasias that stained positively for retinoblastoma (70.33% and 42.14%, respectively; p=0.09; Mann-Whitney-U test). There were no significant differences between K-ras codon 12 point mutation-positive and -negative endometrial carcinomas regarding the proliferative activity of the cancer (mean Indexes 47.6% and 43.8%, respectively; p=0.66, Mann-Whitney-U test). Our data support the view that MIB-1 proliferative activity was significantly increased with a decrease of the histological grading and with the myometrial invasion of human endometrial cancer.

Expression of the cell-cycle regulatory proteins (pRb, cyclin D1, p16INK4A and cdk4) in human endometrial cancer: correlation with clinicopathological features

AbstractIntroduction. Derailments of the control mechanisms in the G1/S phase of the cell cycle play a fundamental role in the initiation and progression of cancer. However, only a few reports have addressed the issue of simultaneously occurring abnormalities of Rb-pathway components in malignant endometrial tumors.Methods. Currently, we assessed the expression of cell-cycle regulatory proteins (pRb, cyclin D1, p16INK4A and cdk4) in 48 sporadic endometrial cancers, and investigated these tumors for a possible relationship between aberrant protein staining and clinicopathological variables of cancer and RB-LOH.Results. There was abnormal pRb, cyclin D1, p16INK4A and cdk4 immunoreactivity in 2%, 50%, 6% and 25% of cases, respectively. Altogether, 33 of 48 (69%) endometrial malignant tumors showed abnormal expression of at least one Rb-pathway protein immunohistochemically. However, there was significant correlation neither between the cell-cycle regulators nor between the frequency of pRb, p16INK4A and cyclin D1 abnormalities and clinicopathological variables of cancer, but a significant correlation did exist between cdk4 staining and the clinical stage of disease (P<0.05, Fishers exact test). Moreover, an inverse relationship was also demonstrated between cdk4 expression and patient age (r=-0.367; P=0.01). However, none of the cell-cycle regulatory proteins, except for pRb, was related to loss of heterozygosity at locus 13q14.Conclusion. As a conclusion, derailments of the Rb-pathway components, cyclin D1 and cdk4 in particular, seems to participate in the endometrial cancer development in humans. Overexpression of cdk4 was related to the progression of neoplastic disease and corresponds with age of onset, suggesting a major role of altered cdk4 immunoreactivity in the progression of endometrial cancer.