Emelie Karnevi

Metformin-mediated growth inhibition involves suppression of the IGF-I receptor signalling pathway in human pancreatic cancer cells

BackgroundEpidemiological studies have shown direct associations between type 2 diabetes and obesity, both conditions associated with hyperglycaemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin. In this study, the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin were investigated.MethodsThe human pancreatic cancer cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were grown in normal (5 mM) or high (25 mM) glucose conditions, with or without metformin. The influence by metformin on proliferation, apoptosis and the AMPK and IGF-IR signalling pathways were evaluated in vitro.ResultsMetformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions. Hyperglycaemia however, protected against the metformin-induced growth inhibition. The anti-proliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPKThr172 together with an inhibition of the insulin/insulin-like growth factor-I (IGF-I) receptor activation and downstream signalling mediators IRS-1 and phosphorylated Akt. Furthermore, exposure to metformin during normal glucose conditions led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose levels promoted a more robust IGF-I response and Akt activation which correlated to stimulated AMPKSer485 phosphorylation and impaired AMPKThr172 phosphorylation, resulting in reduced anti-proliferative and apoptotic effects by metformin.ConclusionOur results indicate that metformin has direct anti-tumour activities in pancreatic cancer cells involving AMPKThr172 activation and suppression of the insulin/IGF signalling pathways. However, hyperglycaemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile and prevent metformin from fully switching off the growth promoting signals in pancreatic cancer cells.

Tumour-educated macrophages display a mixed polarisation and enhance pancreatic cancer cell invasion.

At the time of diagnosis, almost 80% of pancreatic cancer patients present with new‐onset type 2 diabetes (T2D) or impaired glucose tolerance. T2D and pancreatic cancer are both associated with low‐grade inflammation. Tumour‐associated macrophages (TAMs) have a key role in cancer‐related inflammation, immune escape, matrix remodelling and metastasis. In this study, the interplay between tumour cells and immune cells under the influence of different glucose levels was investigated. Human peripheral blood mononuclear cells were exposed in vitro to conditioned medium from BxPC‐3 human pancreatic cancer cells, in normal (5 mm) or high (25 mm) glucose levels. Flow cytometry analyses demonstrated that tumour‐derived factors stimulated differentiation of macrophages, with a mixed classical (M1‐like) and alternatively activated (M2‐like) phenotype polarisation (CD11c+CD206+). High‐glucose conditions further enhanced the tumour‐driven macrophage enrichment and associated interleukin (IL)‐6 and IL‐8 cytokine levels. In addition, hyperglycaemia enhanced the responsiveness of tumour‐educated macrophages to lipopolysaccharide, with elevated cytokine secretion compared with normal glucose levels. Tumour‐educated macrophages were found to promote pancreatic cancer cell invasion in vitro, which was significantly enhanced at high glucose. The anti‐diabetic drug metformin shifted the macrophage phenotype polarisation and reduced the tumour cell invasion at normal, but not high, glucose levels. In conclusion, this study demonstrates that pancreatic cancer cells stimulate differentiation of macrophages with pro‐tumour properties that are further enhanced by hyperglycaemia. These findings highlight important crosstalk between tumour cells and TAMs in the local tumour microenvironment that may contribute to disease progression in pancreatic cancer patients with hyperglycaemia and T2D.

Impact by pancreatic stellate cells on epithelial-mesenchymal transition and pancreatic cancer cell invasion: Adding a third dimension in vitro

Pancreatic cancer is associated with a highly abundant stroma and low-grade inflammation. In the local tumour microenvironment, elevated glucose levels, the presence of tumour-associated stellate cells and macrophages are hypothesised to promote the tumour progression and invasion. The present study investigated the influence by the microenvironment on pancreatic cancer cell invasion in vitro. After co-culture with tumour-associated pancreatic stellate cells (TPSCs), pancreatic cancer cells displayed up to 8-fold reduction in levels of epithelial-mesenchymal transition (EMT) markers E-cadherin and ZO-1, while β-catenin and vimentin levels were increased. A 3D organotypic model showed that TPSCs stimulated pancreatic cancer cell invasion, both as single cell (PANC-1) and cohort (MIAPaCa-2) invasion. The combined presence of TPSCs and M2-like macrophages induced invasion of the non-invasive BxPC-3 cells. High glucose conditions further enhanced changes in EMT markers as well as the cancer cell invasion. In summary, co-culture with TPSCs induced molecular changes associated with EMT in pancreatic cancer cells, regardless of differentiation status, and the organotypic model demonstrated the influence of microenvironmental factors, such as glucose, stellate cells and macrophages, on pancreatic cancer cell invasion.

Expression and Prognostic Significance of Human Epidermal Growth Factor Receptors 1 and 3 in Gastric and Esophageal Adenocarcinoma

Background Gastric and esophageal adenocarcinomas are major global cancer burdens. These cancer forms are characterized by a poor prognosis and a modest response to chemo- radio- and targeted treatment. Hence there is an obvious need for further enhanced diagnostic and treatment strategies. The aim of this study was to examine the expression and prognostic impact of human epidermal growth factor receptor 1 (HER1/EGFR) and 3 (HER3), as well as the occurrence of EGFR and KRAS mutations in gastric and esophageal adenocarcinoma. Methods Immunohistochemical expression of EGFR and HER3 was analysed in all primary tumours and a subset of lymph node metastases in a consecutive cohort of 174 patients with adenocarcinoma of the stomach, cardia and esophagus. The anti-HER3 antibody used was validated by siRNA-mediated knockdown, immunohistochemistry and quantitative real-time PCR. EGFR and KRAS mutation status was analysed by pyrosequencing tecchnology. Results and Discussion High EGFR expression was an independent risk factor for shorter overall survival (OS), whereas high HER3 expression was associated with a borderline significant trend towards a longer OS. KRAS mutations were present in only 4% of the tumours and had no prognostic impact. All tumours were EGFR wild-type. These findings contribute to the ongoing efforts to decide on the potential clinical value of different HERs and druggable mutations in gastric and esophageal adenocarcinomas, and attention is drawn to the need for more standardised investigational methods.

Expression and prognostic significance of human epidermal growth factor receptors 1, 2 and 3 in oesophageal and gastric adenocarcinomas preneoadjuvant and postneoadjuvant treatment

Aims Neoadjuvant treatment has now become the standard of care for oesophageal and gastric cancer. The aim of this study was to investigate the influence of neoadjuvant therapy on the expression of human epidermal growth factor receptor 1 (HER1/EGFR), HER2 and HER3, in oesophageal and gastric adenocarcinoma. Methods Immunohistochemical expression of EGFR, HER2 and HER3 was examined and compared in pretreatment biopsies, post-treatment surgical resection specimens and metastases in a retrospective cohort of 166 patients with adenocarcinoma of the oesophagus or stomach. The relationship between expression of the investigative markers and histopathological response to neoadjuvant treatment, overall survival (OS) and recurrence free survival (RFS) was analysed. Results Conversion of protein expression between pretreatment biopsy and post-treatment surgical resection was seen in 4.6% of the cases for EGFR, 5.9% for HER2% and 19.4% for HER3. Histopathological response to neoadjuvant treatment was significantly and stepwise associated with OS and RFS . High HER3 protein expression in post-treatment surgical resection specimens was significantly associated with a prolonged OS in univariable analysis (HR=0.39; 95% CI 0.17 to 0.93), but did not remain significant in multivariable analysis. Expression of EGFR and HER2 in post-treatment surgical resection specimens was not prognostic. No correlation between pretreatment HER-protein expression and histopathological response was seen. Conclusions The results from this study underscore the need for further studies on the influence of neoadjuvant treatment on biomarker expression, as this may influence treatment strategy as well as prognosis. Histopathological response is validated as a useful prognostic factor.

Translational study reveals a two-faced role of RBM3 in pancreatic cancer and suggests its potential value as a biomarker for improved patient stratification

Periampullary adenocarcinoma, including pancreatic cancer, is a heterogeneous group of tumors with dismal prognosis, partially due to lack of reliable targetable and predictive biomarkers. RNA-binding motif protein 3 (RBM3) has previously been shown to be an independent prognostic and predictive biomarker in several types of cancer. Herein, we examined the prognostic value of RBM3 in periampullary adenocarcinoma, as well as the effects following RBM3 suppression in pancreatic cancer cells in vitro. RBM3 mRNA levels were examined in 176 pancreatic cancer patients from The Cancer Genome Atlas. Immunohistochemical expression of RBM3 was analyzed in tissue microarrays with primary tumors and paired lymph node metastases from 175 consecutive patients with resected periampullary adenocarcinoma. Pancreatic cancer cells were transfected with anti-RBM3 siRNA in vitro and the influence on cell viability following chemotherapy, transwell migration and invasion was assessed. The results demonstrated that high mRNA-levels of RBM3 were significantly associated with a reduced overall survival (p = 0.026). RBM3 protein expression was significantly higher in lymph node metastases than in primary tumors (p = 0.005). High RBM3 protein expression was an independent predictive factor for the effect of adjuvant chemotherapy and an independent negative prognostic factor in untreated patients (p for interaction = 0.003). After siRNA suppression of RBM3 in vitro, pancreatic cancer cells displayed reduced migration and invasion compared to control, as well as a significantly increased resistance to chemotherapy. In conclusion, the strong indication of a positive response predictive effect of RBM3 expression in pancreatic cancer may be highly relevant in the clinical setting and merits further validation.

Abstract B17: Tumor-associated stellate cells promote an invasive phenotype of pancreatic cancer cells

At the time of diagnosis, almost 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance. Pancreatic cancer is associated with a highly abundant stroma, which is believed to influence the pathogenesis of the disease. In the local tumor microenvironment, both elevated glucose levels and the presence of tumor-associated stellate cells are hypothesized to promote the tumor progression and invasion by tumor cells. In this study, the influence by the microenvironment on pancreatic cancer cell invasion was investigated in vitro. Epithelial-to-mesenchymal transition (EMT) of human pancreatic cancer cells co-cultured in normal (5mM) or high (25 mM) glucose conditions with tumor-associated pancreatic stellate cells (TPSCs) was monitored by Western immunoblotting. In addition, a three-dimensional organotypic model was established to study and visualize the pancreatic cancer cell invasion. After co-culture, the expression of several EMT-markers was altered. The levels of E-cadherin were reduced by more than 95% ( P P Citation Format: Emelie Karnevi, Ann H. Rosendahl, Moin A. Saleem, Roland Andersson. Tumor-associated stellate cells promote an invasive phenotype of pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B17.

Abstract 5156: Hyperglycemia enhances IGF-I responses and alters the AMPK activation profile that protects against metformin-induced growth inhibition in pancreatic cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epidemiological studies have shown direct associations between type 2 diabetes (T2D) and obesity, both conditions associated with hyperglycemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients are diagnosed with either impaired glucose tolerance or new-onset T2D one or two years prior to their cancer diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin, an insulin-sensitizing and glucose lowering drug. We investigated the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin. We found that metformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions, but that hyperglycemia protected against the metformin-induced growth inhibition. The antiproliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPK(T172) together with a dose-dependent suppression of the insulin/insulin-like growth factor (IGF) signaling mediators IRS-1 and phosphorylated AKT. Furthermore, exposure to metformin during normal glucose led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose promoted a more robust IGF-I response and AKT activation, that stimulated AMPK(S485) phosphorylation and suppressed AMPK(T172) phosphorylation resulting in reduced antiproliferative and apoptotic effects by metformin. In conclusion, our results indicate that metformin has direct antitumor activities in pancreatic cancer cells involving AMPK(T172) activation and modulation of the insulin/IGF signaling pathways. However, hyperglycemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile that prevent metformin from fully switching of the growth promoting signals in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5156. doi:1538-7445.AM2012-5156