Mariusz Koda

Increased expression of leptin and the leptin receptor as a marker of breast cancer progression : Possible role of obesity-related stimuli

Purpose: Recent in vitro studies suggested that the autocrine leptin loop might contribute to breast cancer development by enhancing cell growth and survival. To evaluate whether the leptin system could become a target in breast cancer therapy, we examined the expression of leptin and its receptor (ObR) in primary and metastatic breast cancer and noncancer mammary epithelium. We also studied whether the expression of leptin/ObR in breast cancer can be induced by obesity-related stimuli, such as elevated levels of insulin, insulin-like growth factor-I (IGF-I), estradiol, or hypoxic conditions. Experimental Design: The expression of leptin and ObR was examined by immunohistochemistry in 148 primary breast cancers and 66 breast cancer metastases as well as in 90 benign mammary lesions. The effects of insulin, IGF-I, estradiol, and hypoxia on leptin and ObR mRNA expression were assessed by reverse transcription-PCR in MCF-7 and MDA-MB-231 breast cancer cell lines. Results: Leptin and ObR were significantly overexpressed in primary and metastatic breast cancer relative to noncancer tissues. In primary tumors, leptin positively correlated with ObR, and both biomarkers were most abundant in G3 tumors. The expression of leptin mRNA was enhanced by insulin and hypoxia in MCF-7 and MDA-MB-231 cells, whereas IGF-I and estradiol stimulated leptin mRNA only in MCF-7 cells. ObR mRNA was induced by insulin, IGF-I, and estradiol in MCF-7 cells and by insulin and hypoxia in MDA-MB-231 cells. Conclusions: Leptin and ObR are overexpressed in breast cancer, possibly due to hypoxia and/or overexposure of cells to insulin, IGF-I, and/or estradiol.

Increased expression of connexins 26 and 43 in lymph node metastases of breast cancer

Background: Gap junctions are intercellular channels composed of connexins, which mediate the direct passage of small molecules between neighbouring cells. They are involved in regulation of cell cycle, cell signalling, and differentiation, and probably invasion and metastasis. The role of connexins in the metastatic process is controversial, because some studies indicate that connexin expression is inversely correlated with metastatic capacity. In contrast, others demonstrate that connexins may be involved in metastasis. In addition, connexin status in breast cancer metastasis has not been widely studied. Methods: We evaluated by immunohistochemistry the expression of connexin 26 (Cx26) and connexin 43 (Cx43) in primary breast tumours (PTs) and matched paired metastases to lymph nodes (MLNs). Results: In PTs, we observed predominantly cytoplasmic localisation of evaluated connexins, indicating alterations in connexin expression in breast cancer cells. We demonstrated that expression of Cx26 and Cx43 was increased in MLNs compared with PTs (p<0.00001 and p<0.001, for CX26 and Cx43, respectively). In addition, Cx26 and Cx43 negative PTs developed Cx26 and Cx43 positive MLNs. Furthermore, besides increased cytoplasmic staining, enhanced membranous localisation of Cx43, typical of normal cells, was found in MLNs. Additionally, membranous Cx26 expression appeared only in metastatic breast cancer cells. Conclusions: These findings suggest that connexins may contribute to the efficient metastasising of breast cancer to the lymph nodes.

Overexpression of the obesity hormone leptin in human colorectal cancer

Background: Leptin is an adipocyte-derived neurohormone, high levels of which are found in obese individuals. Leptin controls energy expenditure, acting in the brain, and regulates different processes in peripheral organs. Recent studies have suggested that leptin may be involved in cancer development and progression. Aims: To analyse leptin expression in human colorectal cancer as well as in colorectal mucosa and colorectal adenomas. Methods: Leptin expression was assessed by immunohistochemistry in 166 colorectal cancers, 101 samples of colorectal mucosa and 41 adenomas. Leptin concentration in colorectal cancer was correlated with selected clinicopathological features. Results: Immunoreactivity for leptin was observed in 51.2% (85/166) of primary colorectal cancers. In adenomas leptin expression was observed in 14.6% (6/41) of studied cases. In normal mucosa, leptin was present at low levels, except in tumour bordering areas where its concentration appeared to reflect levels in the adjacent cancer tissue. Leptin expression in colorectal cancer significantly correlated with tumour G2 grade (p = 0.002) as well as with histological type (adenocarcinoma) of tumours (p = 0.044). Conclusions: Results indicate that leptin is overexpressed in human colorectal cancer, which suggests that the hormone might contribute to colorectal cancer development and progression.

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)

Expression of insulin receptor substrate 1 in primary breast cancer and lymph node metastases

Background: Insulin receptor substrate 1 (IRS-1) transmits signals from the insulin-like growth factor I receptor (IGF-IR) and insulin receptor (IR) and has been associated with the pathogenesis of cancer. IRS-1 downregulation has been suggested to play a role in breast cancer progression, but no simultaneous assessments of IRS-1 expression in primary breast cancer and metastases have been performed. Aims: To assess IRS-1 expression in primary and metastatic breast cancer. Methods: IRS-1 expression was analysed by means of immunohistochemistry in 109 samples of primary breast cancer and in 42 matched primary and metastatic tumours. In addition, IRS-1 expression was correlated with selected clinicopathological features, including oestrogen receptor α (ERα) and proliferation marker Ki-67 status. Results: Positive cytoplasmic IRS-1 immunostaining was found in 69.7% (76 of 109) and 76.2% (32 of 42) of the primary and metastatic tumours, respectively. Both IRS-1 positive and IRS-1 negative primary tumours produced IRS-1 positive and IRS-1 negative metastases. IRS-1 expression in primary tumours correlated with poorly differentiated (G3) breast cancer (p<0.005) and with lymph node involvement (p<0.05). In the subgroup of ERα positive primary tumours, IRS-1 expression positively correlated with Ki-67 (p<0.02, r = 0.351), but in the subgroup of ERα negative primary tumours there was a negative correlation (p<0.03, r = −0.509). IRS-1 expression in lymph node metastases correlated with neither ERα nor Ki-67. Conclusions: IRS-1 might be involved in breast cancer progression. Knowledge about differences between primary and metastatic tumours might help to understand mechanisms of breast cancer progression and lead to the development of more effective anticancer drugs.

Alterations in Connexin26 Expression during Colorectal Carcinogenesis

Background: Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions, which consist of transmembrane proteins called connexins (Cxs). Human colorectal epithelial cells express Cx32 and Cx43. Methods: Tissue samples (152 from colorectal cancer and 75 from adenoma) were investigated by immunohistochemistry, using the antibody for Cx26. Moreover, Cx26 was assessed in normal epithelium of the colon and rectum, adjacent to colorectal cancer. Results: In normal epithelium and adenomas, intercellular, punctate staining for Cx26 was observed. In adenomas with severe dysplasia, focally decreased expression of Cx26 was observed. Among 152 colorectal cancers, 55.9% classified only as adenocarcinoma stained positive for Cx26, but mainly cytoplasmic staining was found. We observed a positive correlation between Cx26 expression and tumor G2 grade (p < 0.005). The expression of Cx26 did not correlate with age, sex of patients, tumor localization, lymph node status or tumor size. Conclusions: Our results show that during colorectal carcinogenesis, loss of normal intercellular Cx expression occurs. Furthermore, the cytoplasmic presence of Cx26 could indicate a different role of Cx26 in neoplastic cells than participation in GJIC.

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.

Relationships between hypoxia markers and the leptin system, estrogen receptors in human primary and metastatic breast cancer: effects of preoperative chemotherapy

BackgroundTumor hypoxia is marked by enhanced expression of hypoxia-inducible factor-α (HIF-1α) and glucose transporter-1 (Glut-1). Hypoxic conditions have also been associated with overexpression of angiogenic factors, such as leptin. The aim of our study was to analyze the relationships between hypoxia markers HIF-1α, Glut-1, leptin, leptin receptor (ObR) and other breast cancer biomarkers in primary and metastatic breast cancer in patients treated or untreated with preoperative chemotherapy.MethodsThe expression of different biomarkers was examined by immunohistochemistry in 116 primary breast cancers and 65 lymph node metastases. Forty five of these samples were obtained form patients who received preoperative chemotherapy and 71 from untreated patients.ResultsIn primary tumors without preoperative chemotherapy, HIF-1α and Glut-1 were positively correlated (p = 0.02, r = 0.437). HIF-1α in primary and metastatic tumors without preoperative therapy positively correlated with leptin (p < 0.0001, r = 0.532; p = 0.013, r = 0.533, respectively) and ObR (p = 0.002, r = 0.319; p = 0.083, r = 0.387, respectively). Hypoxia markers HIF-1α and Glut-1 were negatively associated with estrogen receptor alpha (ERα) and positively correlated with estrogen receptor beta (ERβ). In this group of tumors, a positive correlation between Glut-1 and proliferation marker Ki-67 (p = 0.017, r = 0.433) was noted. The associations between HIF-1α and Glut-1, HIF-1α and leptin, HIF-1α and ERα as well as Glut-1 and ERβ were lost following preoperative chemotherapy.ConclusionsIntratumoral hypoxia in breast cancer is marked by coordinated expression of such markers as HIF-1α, Glut-1, leptin and ObR. The relationships among these proteins can be altered by preoperative chemotherapy.

Expression of the Insulin‐Like Growth Factor‐I Receptor and Proapoptotic Bax and Bak Proteins in Human Colorectal Cancer

Abstract: Insulin‐like growth factor‐I (IGF‐I) and IGF‐I receptor (IGF‐IR), despite their well‐known roles in cell survival and proliferation, can also weakly enhance apoptosis. To study the relationships between the IGF‐IR and Bax as well as Bak, 144 cases of colorectal cancer were examined by immunohistochemistry, using the avidin‐biotin‐peroxidase method. The results were correlated with selected clinicopathological features of colorectal cancer and with the expression of IGF‐IR, Bax, and Bak in normal colon mucosa. In Bax‐, Bak‐, or IGF‐IR‐positive cancers, the adjacent colorectal mucosa revealed positive immunostaining for these proteins. In the majority of Bax‐, Bak‐, or IGF‐IR‐negative tumors, we observed no staining for these proteins in adjacent mucosa. The strong immunostaining for IGF‐IR, Bax, and Bak was noted in 50.8, 55.5, and 49.3% of tumors, respectively. We observed positive correlations between IGF‐IR and Bax (P < 0.002, r= 0.302), between IGF‐IR and Bak (P < 0.0001, r= 0.407), and between Bax and Bak (P < 0.0001, r= 0.474). No relationship was noted between IGF‐IR expression and tumor grade, stage, or lymph node status. We found negative associations between Bax, Bak, and tumor grade (P < 0.01 and P < 0.003, respectively), but no relationships between Bax and Bak and tumor stage or between Bax and Bak and lymph node status. Our results demonstrate that, in addition to overexpressed IGF‐IR, there are relationships between IGF‐IR and proapoptotic proteins in colorectal carcinomas that could contribute to increased cell turnover and the progression of colorectal cancer.