Andrzej Wincewicz

Expression of Leptin, Leptin Receptor, and Hypoxia‐Inducible Factor 1α in Human Endometrial Cancer

Abstract:  Recent studies suggested that Ob (Ob) and its receptor (ObR) could be involved in the pathogenesis of various human malignancies, among others in endometrial cancer. Moreover, hypoxia, which is associated with solid tumors, might stimulate, through hypoxia‐inducible factor 1α (HIF‐1α), expression of Ob and ObR. In this article, we analyzed by immunohistochemistry the expression of Ob, ObR, and HIF‐1α in 60 cases of human endometrial cancer tissues as well as in 25 cases of normal endometria. Additionally, we assessed correlations among studied proteins as well as relationships with selected clinicopathological features of endometrial cancer. Immunoreactivity for Ob, ObR, and HIF‐1α protein was observed in 56.7%, 30.0%, and 78.3% of endometrial cancers, respectively. The expression of HIF‐1α showed a significant positive correlation with Ob (P < 0.0001, r= 0.573) and ObR (P= 0.020, r= 0.299). Moreover, we noted positive correlation between Ob and ObR (P= 0.001, r= 0.429). No statistically significant relationship was revealed between Ob, ObR, and HIF‐1α protein in regard to patients age, histological grade, and extent of tumor growth (pT). In conclusion, HIF‐1α, which is related to tissue hypoxia in endometrial cancer, seems to be associated with overexpression of Ob and ObR. Ob could exert autocrine effect to stimulate endometrial cancer progression. Thus the autocrine Ob loop should be taken into consideration as a novel potential target in endometrial cancer prevention and treatment.

Leptin - From Regulation of Fat Metabolism to Stimulation of Breast Cancer Growth

Leptin restricts intake of calories as a satiety hormone. It probably stimulates neoplastic proliferation in breast cancer, too. Growth of malignant cells could be regulated by various leptininduced second messengers like STAT3 (signal transducers and activators of transcription 3), AP-1 (transcription activator protein 1), MAPK (mitogenactivated protein kinase) and ERKs (extracellular signalregulated kinases). They seem to be involved in aromatase expression, generation of estrogens and activation of estrogen receptor α (ERα) in malignant breast epithelium. Leptin may maintain resistance to antiestrogen therapy. Namely, it increased activation of estrogen receptors,therefore,it was suspected to reduce or even overcome the inhibitory effect of tamoxifen on breast cell proliferation. Although several valuable reviews have been focused on the role of leptin in breast cancer,the status of knowledge in this field changes quickly and our insight should be continuously revised. In this summary,we provide refreshed interpretation of intensively reported scientific queries of the topic.(Pathology Oncology Research Vol 12, No 2, 69–72)

Clinicopathological significance and linkage of the distribution of HIF-1α and GLUT-1 in human primary colorectal cancer

HIF-la induces GLUT-1 expression, and their presence has been evaluated in colorectal cancer. However, the expressions of GLUT-1 and HIF-lα have not been investigated together with reference to clinicopathological characteristics in human colorectal cancer. The aim of our study was to compare the expression of HIF-lα and GLUT-1 with various clinicopathological features of colorectal cancer. The presence of HIF-lα and GLUT-1 was visualized immunohistochemically in 123 primary tumors. Membranous localization of GLUT-1 was found in multifocally necrotizing cancer samples, while pure cytoplasmic perinuclear, mostly supranuclear GLUT-1 accumulation was characteristic of cancer fields with lack of necrosis. HIF-la was located in the cytoplasm and occasionally in the nuclei of cancer cells. Immunoreactivity to GLUT-1 was significantly higher in node-positive cancers compared with nodenegative ones (p=0.04), confirming our earlier results obtained on a larger number of patients. Non-mucinous adenocarcinomas expressed GLUT-1 and HIF-lα with significantly greater frequency than mucinous adenocarcinomas (p=0.002, p=0.0002, respectively). GLUT-1 and HIF-la expression did not differ in relation to tumor stage, location, or patients′ age or gender. In contrast to that of GLUT-1, expression of HIF-lα correlated with grade (p=0.00003) without difference with regard to pN status. HIF-lα expression correlated with GLUT-1 expression in the whole patient population, as well as in all clinicopathological groups except for the pTl+pT2 group. Although the coexpression of cytoplasmic HIF-la and GLUT-1 does not directly prove the dependence between HIF-1 as a nuclear transcriptional factor and GLUT-1 as its downstream protein, it is evidence of their simultaneous upregulation. The extranuclear accumulation of HIF-la and GLUT-1 requires further studies to explain its significance in colorectal cancer.

Relations of TGF-β1 with HIF-1α, GLUT-1 and longer survival of colorectal cancer patients

Aims and Methods: During colorectal carcinogenesis, transforming growth factor beta 1 (TGF‐β1) undergoes a functional change from suppression of cancer cell proliferation to inhibition of T cell mediated anti‐cancer immunity. We aimed to evaluate relations among TGF‐β1 and cancer cell survival factors hypoxia inducible factor‐1 alpha (HIF‐1α) and glucose transporter 1 (GLUT‐1) by immunohistochemistry in 108 colorectal cancers. Results: TGF‐β1 was detected in 87% (94/108), HIF‐1α in 85% (92/108), and GLUT‐1 in 65% (70/108) of colorectal cancers. Not only did TGF‐β1 accumulate in cytoplasm of cancer cells but also there was strong immunoreactivity to TGF‐β1 in adjacent inflammatory cells. GLUT‐1 was visualised in a membranous fashion while HIF‐1 was expressed in a paranuclear pattern and occasionally in nuclei of malignant cells. Cancer immunoreactivities to TGF‐β1 correlated with HIF‐1α (p < 0.001, r = 0.516) and GLUT‐1 (p > 0.001, r = 0.355) in general and subgroups of different clinicopathological traits. TGF‐β1 expressions of inflammatory infiltrates correlated with longer patient survival (p = 0.05, r = 0.449) and immunoreactivities to HIF‐1α of cancer cells (p = 0.008, r = 0.254) particularly in node positive and deeply invading cancers but failed to associate significantly with GLUT‐1. Conclusions: HIF‐1α and GLUT‐1 could cooperate with TGF‐β1, and TGF‐β1 might mediate cross‐talk between the inflammatory environment and tumour with a favourable impact on patient survival.

Insulin-like growth factor-I receptor correlates with connexin 26 and Bcl-xL expression in human colorectal cancer.

Abstract:  Insulin‐like growth factor (IGF) and its receptor (IGF‐IR) play an important role in mitogenesis, apoptosis, growth, and proliferation of several types of cancers. Overexpression of IGF‐IR in colorectal cancer is associated with increase of cancer cell proliferation and migration as well as inhibition of apoptosis. In our previous reports we demonstrated correlations between IGF‐IR and apoptosis. Moreover, we observed relationships between connexin26 (Cx26) expression and apoptotic markers in human colorectal cancer. Recently, it has been shown that expression of connexins and gap junction (GJ) functions are also regulated by growth factors, including IGF‐I. Therefore, in this study we have focused on the relationships between IGF‐IR and Cx26 as well as Bcl‐xL expression. A total number of 115 cases of colorectal cancer were examined by immunohistochemistry, using the avidin‐biotin‐peroxidase method. Associations among the above proteins were assessed in the entire group of colorectal cancer patients and its subgroups, depending on lymph node involvement (N0 and N1), histological grade (G2 and G3), extent of tumor growth (pT1 + pT2 and pT3 + pT4), histopathologic type (adenocarcinoma and mucinous carcinoma), sex, age (≤60 and >60), and tumor site (colon and rectum). The expression of IGF‐IR, Cx26, and Bcl‐xL was noted in 47%, 56.5%, and 75.6% of the tumors, respectively. In the entire group of patients we found a positive correlation between IGF‐IR and Cx26 ( P < 0.0001, r = 0.374) as well as between IGF‐IR and Bcl‐xL ( P < 0.0001, r = 0.344). Our results may suggest that the insulin‐like growth system is involved in regulation of apoptosis and probably connexin expression in colorectal cancer cells.

Aberrant Distributions and Relationships Among E-cadherin, β-catenin, and Connexin 26 and 43 in Endometrioid Adenocarcinomas

During carcinogenesis, loss of intracellular cohesion is observed among cancer cells with altered expression of such adhesion molecules as E-cadherin and β-catenin, and aberrant expression and cellular location of intercellular gap junction proteins-connexins. The aim of this study was to evaluate immunohistochemically the expression and relationship between E-cadherin and β-catenin, and the connexins Cx26 and Cx43 in 86 endometrioid adenocarcinomas. The aberrant cytoplasmic translocation of the studied proteins was a predominant finding, whereas only a minority of cases showed normal, nuclear β-catenin labeling or membranous distribution of the remaining molecules. E-cadherin was positively and significantly associated with β-catenin (P=0.001, r=0.366), as was Cx26 with Cx43 (P<0.001, r=0.719), E-cadherin with Cx26 (P<0.001, r=0.413), and E-cadherin and Cx43 (P<0.001, r=0.434) in all cancers. A subgroup of endometrioid adenocarcinomas (FIGO IB+II) exclusively showed a positive significant association between the expression of β-catenin and Cx26 (P=0.038, r=0.339). In addition, there were significantly more β-catenin-positive carcinomas among superficially spreading cancers (FIGO IA) than among deeper invading neoplasms (FIGO IB+II) (P=0.056). The altered location of the studied proteins indicates impairment of their physiological functions. In particular, normal membranous distribution of E-cadherin and connexins is lost and replaced by abnormal cytoplasmic accumulation in most cancers, and thus intercellular ties are expected to be weakened and loosened as a consequence. In contrast, the lack of relationship between β-catenin and connexins, E-cadherin seems to be closely associated with the expression of Cx26 and Cx43 in endometrioid adenocarcinomas.

STAT3 and hypoxia induced proteins--HIF-1alpha, EPO and EPOR in relation with Bax and Bcl-xL in nodal metastases of ductal breast cancers.

STAT3 contributes to increase of EPO expression which is also HIF-1 dependent. EPO receptor activates STAT3. Expressions of STAT3 and hypoxia induced proteins: HIF-1, EPO and EPOR show mutual correlations in primary ductal breast cancers, which suggest co-operation among these proteins. Moreover, EPO-EPOR signaling was reported to mediate cell survival by targeting Bcl-xL in competition with Bax-dependent apoptosis. Our present study was focused on immunohistochemical evaluation of STAT3, HIF-1alpha, EPO and EPOR in relation to apoptosis regulators, Bax and Bcl-xL in 39 metastases of ductal breast cancers to lymph nodes. The proteins were abundantly expressed by cancer cells. HIF-1alpha correlated with EPOR in all and in chemotherapy treated metastases (r=0.428, p=0.007 and r=0.462, p=0.040, respectively). HIF-1 associated significantly with EPO in chemotherapy spared metastases (r=0.549, p=0.015) and comparison between those proteins almost reached statistical significance in entire number of metastatic breast cancers (r=0.309, p=0.056). Metastases from T2 primary tumors had significantly higher expressions of HIF-1alpha, EPO and EPOR compared to T1 originating metastases (p=0.020, p=0.028, p=0.021, respectively). Bax correlated with EPO and EPOR in all studied nodal metastases (r=0.449, p=0.006 and r=0.421, p=0.011, respectively) and so did Bcl-xL with HIF-1alpha (r=0.440, p=0.007), EPO and EPOR (r=0.383, p=0.021, r=0.495, p=0.002, respectively). Metastatic breast cancers seem to be areas of intensive signaling by STAT3, HIF-1, EPO and EPOR. Strong Bax and Bcl-xL labeling reflects accelerated cell turnover in nodal metastases. By means of association with Bcl-xL, HIF-1alpha, EPO and EPOR could favor growth of nodal metastases and survival of breast cancers cells.

Significant Coexpression of GLUT‐1, Bcl‐xL, and Bax in Colorectal Cancer

Abstract:  Hypoxic cancer cells overexpress Glucose transporter 1 (GLUT‐1) to accelerate glucose intake mainly for low effective, anaerobic respiration, so that they would not die of oxygen deficiency. Ischemic cell injury triggers apoptosis. Regulators of cell suicide like Bax and Bcl‐xL combine their functions to cause apoptosis or to rescue cells from death. GLUT‐1, Bax, and Bcl‐xL are of prognostic significance in colorectal cancer but they have not been compared, yet. Thus, we aimed to determine eventual correlations between GLUT‐1, Bax, and Bcl‐xL in association with different clinicopathological features of colorectal cancer patients. Expressions of the proteins were evaluated in specimens of 150 colorectal patients by immunohistochemistry. The levels of tissue expressions were statistically analyzed with Spearmans correlation test. As in group of all the patients, GLUT‐1 matched Bcl‐xL and Bax in statistically significant manner regardless of different node status, grade of histological differentiation, histopathological type, tumor site, gender and age of patients. GLUT‐1 correlated highly with Bcl‐xL in both groups of various tumor growth extent: pT1 + pT2 and pT3 + pT4 tumors (P < 0.016, r= 0.6340, P < 0.0001, r= 0.5204, respectively). Bax correlated with GLUT‐1 (P < 0.0001, r= 0.4284) and Bcl‐xL (P < 0.0001, r= 0.5233) in pT3 and pT4 tumors without any statistical significance in a homologous comparison at pT1 and pT2 stage (P > 0.173, r= 0.1078, P > 0.744, r= 0.1, respectively). Significant coexpression of GLUT‐1, Bcl‐xL, and Bax could point to cooperation of these regulatory proteins in elimination due to irreversible injury, adaptation to hypoxia, reduction of further damage, and survival of colorectal cancer cells.

Expression of Bcl-xL, Bax, and p53 in Primary Tumors and Lymph Node Metastases in Oral Squamous Cell Carcinoma

Abstract:  Disturbances in expression of apoptosis‐associated proteins take part in the development and progression of many human malignancies. The aim of this study was the assessment of correlations among proteins involved in apoptosis—Bcl‐xL, Bax, and p53—as well as relationships of these proteins with selected clinicopathological features in oral squamous cell carcinoma. Consequently, we examined by immunohistochemistry, using the avidin–biotin–peroxidase method, Bcl‐xL, Bax, and p53 expression in 56 samples of primary oral squamous cell carcinoma and in 22 matched pairs of primary and metastatic tumors. The evaluation of immunostaining of Bcl‐xL, Bax, and p53 was analyzed in 10 different tumor fields, and the mean percentage of tumor cells with positive staining was evaluated. The significance of the associations was determined using Spearman correlation analysis and the chi‐square test. We found positive Bcl‐xL, Bax, and p53 immunostaining in 44.6%, 28.6%, and 58.9% of the studied primary tumors and in 63.6%, 45.5%, and 72.7% of lymph node metastases, respectively. Analysis of associations among studied proteins revealed positive correlation between Bcl‐xL and Bax in primary tumors (P < 0.03, r= 0.307). Statistically significant relationship between p53 expression in primary oral cancers and its expression in lymph node metastases (P < 0.02) as well as increased expression of Bcl‐xL, Bax, and p53 in metastatic sites compared with primary tumors could indicate an association of these proteins with oral cancer progression and development of metastases. Moreover, we suppose that knowledge about heterogeneity between primary and metastatic tumor might help to understand mechanisms of oral cancer progression.

Erythropoietin and Erythropoietin Receptor in Colorectal Cancer

Erythropoietin via erythropoietin receptor effectively prevents anemia, giving reasons for a clinical use of erythropoietin in patients with colorectal cancers. However, erythropoietin seems to promote survival of the neoplastic cells in hypoxic environment. The aim of this study was to evaluate immunohistochemically the expression of erythropoietin and erythropoietin receptor in 136 primary colorectal cancers with a correlation to different anatomo-clinical features. Erythropoietin correlated with erythropoietin receptor in colorectal cancers (r = 0.547, P < .00001). Erythropoietin and erythropoietin receptor expressions were statistically higher in adenocarcinomas versus mucinous carcinomas (P = .05 and P = .03, respectively) and in moderately (G2) versus poorly differentiated (G3) tumors (P = .001 and P = .02, respectively). This in vivo study is the first study that provides evidences for the presence of erythropoietin and erythropoietin receptor in human colorectal cancer. The expressions of these proteins strictly depended on grading because the better histological differentiation probably comes from trophic influence of erythropoietin and erythropoietin receptor.