Hui-Hua Chang

High-Fat, High-Calorie Diet Promotes Early Pancreatic Neoplasia in the Conditional KrasG12D Mouse Model

There is epidemiologic evidence that obesity increases the risk of cancers. Several underlying mechanisms, including inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. The goal of the present study was to develop a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, for example, weight gain and metabolic disturbances. Offspring of Pdx-1-Cre and LSL-KrasG12D mice were allocated to either a high-fat, high-calorie diet (HFCD; ∼4,535 kcal/kg; 40% of calories from fats) or control diet (∼3,725 kcal/kg; 12% of calories from fats) for 3 months. Compared with control animals, mice fed with the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of insulin-like growth factor I (IGF-I). The pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells (macrophages and T cells), elevated levels of several cytokines and chemokines, increased stromal fibrosis, and more advanced PanIN lesions. Our results show that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. This model and findings will provide the basis for more robust studies attempting to unravel the mechanisms underlying the cancer-promoting properties of obesity, as well as to evaluate dietary- and chemopreventive strategies targeting obesity-associated pancreatic cancer development. Cancer Prev Res; 6(10); 1064–73. ©2013 AACR.

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice

Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.

Robust Early Inflammation of the Peripancreatic Visceral Adipose Tissue During Diet-Induced Obesity in the KrasG12D Model of Pancreatic Cancer.

Objectives Obesity increases the incidence of multiple types of cancer. Our previous work has shown that a high-fat, high-calorie diet (HFCD) leads to visceral obesity, pancreatic inflammation, and accelerated pancreatic neoplasia in KrasG12D (KC) mice. In this study, we aimed to investigate the effects of an HFCD on visceral adipose inflammation with emphasis on potential differences between distinct visceral adipose depots. Methods We examined the weight and visceral obesity in both wild-type and KC mice on either control diet (CD) or HFCD. After 3 months, mice were killed for histological examination. Multiplex assays were also performed to obtain cytokine profiles between different adipose depots. Results Both wild-type and KC mice on an HFCD exhibited significantly increased inflammation in the visceral adipose tissue, particularly in the peripancreatic fat (PPF), compared with animals on a CD. This was associated with significantly increased inflammation in the pancreas. Cytokine profiles were different between visceral adipose depots and between mice on the HFCD and CD. Conclusions Our results clearly demonstrate that an HFCD leads to obesity and inflammation in the visceral adipose tissue, particularly the PPF. These data suggest that obesity-associated inflammation in PPF may accelerate pancreatic neoplasia in KC mice.

Insulin promotes proliferation and fibrosing responses in activated pancreatic stellate cells.

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high-fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia, and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared with normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 mo similarly displayed increasing peri-islet fibrosis compared with mice fed control diet. Both quiescent and activated PaSC coexpress insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling, and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data support the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.

Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA- and EP1/Ca2+-mediated mechanism in the human pancreatic carcinoma cell line PANC-1.

Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. Proinflammatory prostaglandin E2 (PGE2) and elevated insulin-like growth factor type 1 (IGF-1), related to insulin resistance, are shown to play critical roles in pancreatic cancer progression. We aimed to explore a potential cross talk between PGE2 signaling and the IGF-1/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In PANC-1 human pancreatic cancer cells, we showed that PGE2 stimulated mTORC1 activity independently of Akt, as evaluated by downstream signaling events. Subsequently, using pharmacological and genetic approaches, we demonstrated that PGE2-induced mTORC1 activation is mediated by the EP4/cAMP/PKA pathway, as well as an EP1/Ca(2+)-dependent pathway. The cooperative roles of the two pathways were supported by the maximal inhibition achieved with the combined pharmacological blockade, and the coexistence of highly expressed EP1 (mediating the Ca(2+) response) and EP2 or EP4 (mediating the cAMP/PKA pathway) in PANC-1 cells and in the prostate cancer line PC-3, which also robustly exhibited PGE2-induced mTORC1 activation, as identified from a screen in various cancer cell lines. Importantly, we showed a reinforcing interaction between PGE2 and IGF-1 on mTORC1 signaling, with an increase in IL-23 production as a cellular outcome. Our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca(2+) signaling, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects.

Suppression of Gingival NK Cells in Precancerous and Cancerous Stages of Pancreatic Cancer in KC and BLT-Humanized Mice

The aim of our studies is to determine the dynamics of natural killer (NK) cell modulation in gingivae in precancerous and cancerous stages of pancreatic and oral cancers in P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation and BLT-humanized mice. Wild type and KC mice fed with control diet (CD) or high-fat calorie diet (HFCD), and the pancreatic and oral tumor-bearing humanized BLT (hu-BLT) mice were used to determine precancerous and cancer induced changes in numbers and function of gingival NK cells. Increased numbers of PanIN lesions and the greatest score of inflammation in pancreas of KC mice fed with CD and HFCD co-related with significant decline in percentages of circulating and gingival NK cells, lack of DX5+ NK expansion and increased secretion of IFN-γ and IL-6 after culture. At the malignant stage of pancreatic cancer, hu-BLT tumor-bearing mice had the lowest secretion of IFN-γ from cells dissociated from the gingival tissues as compared to those from non-tumor-bearing mice. Injection of NK cells into tumor-bearing mice increased IFN-γ secretion, and the secretion was similar or higher than those obtained by gingival cells from non-tumor-bearing hu-BLT control mice. The highest increase in IFN-γ secretion was observed when tumor-bearing mice were fed with AJ2 probiotic bacteria and injected with the NK cells. Along with an increase in secretion of IFN-γ, injection of NK cells in the presence and absence of feeding with AJ2 in pancreatic tumor-bearing mice increased percentages of CD45+ and CD3+ T cells in oral gingival cells. Similar results were observed with oral tumors. In conclusion, these results indicated that oral cavity may mirror systemic disease and provide a rationale for why cancer patients may be prone to suffer from diverse oral pathologies.

E-cadherin expression in obesity-associated, Kras-initiated pancreatic ductal adenocarcinoma in mice

BACKGROUND The epithelial-mesenchymal transition (EMT) is critical in the development of invasive epithelial malignancies. EMT is accelerated by inflammation and results in decreased E-cadherin expression. Diet-induced obesity is an inflammatory state that accelerates pancreatic carcinogenesis; its effect on EMT and E-cadherin expression in the development of pancreatic ductal adenocarcinoma is unclear. METHODS Conditional Kras(G12D) mice were fed a control diet or a high-fat, high-calorie diet for 3 or 9 months (n = 10 each). Immunohistochemistry with anti-E-cadherin antibody was performed. E-cadherin expression was characterized by staining intensity, location, and proportion of positive cells. In vitro expression of E-cadherin and Slug in primary pancreatic intraepithelial neoplasia (PanIN) and cancer cells was determined by Western blot. RESULTS The HFCD led to increased weight gain in both 3- (15.8 vs 5.6 g, P < .001) and 9-month (19.8 vs 12.9 g, P = .007) mice. No differences in E-cadherin expression among various stages of preinvasive PanIN lesions were found--regardless of age or diet. In invasive cancer, E-cadherin expression was aberrant, with loss of membranous staining and prominent cytoplasmic staining, associated with strong, cytoplasmic expression of β-catenin. In vitro expression of E-cadherin was greatest in primary PanIN cells, accompanied by absent Slug expression. Cancer cell lines demonstrated significantly decreased E-cadherin expression in the presence of upregulated Slug. CONCLUSION Despite increased pancreatic inflammation and accelerated carcinogenesis, the high-fat, high-calorie diet did not induce changes in E-cadherin expression in PanIN lesions of all stages. Invasive lesions demonstrated aberrant cytoplasmic E-cadherin staining. Loss of normal membranous localization may reflect a functional loss of E-cadherin.

Direct growth-inhibitory effects of prostaglandin E2 in pancreatic cancer cells in vitro through an EP4/PKA-mediated mechanism

Background: There is strong evidence linking inflammation and the development of pancreatic ductal adenocarcinoma. Cyclooxygenase‐2 (COX‐2) and COX‐2‐derived PGE2 are overexpressed in human and murine pancreatic ductal adenocarcinoma. Several studies have demonstrated an important role of COX‐2‐derived PGE2 in tumor‐stroma interactions; however, the direct growth effects of prostaglandin E2 (PGE2) on pancreatic ductal adenocarcinoma cells is less well defined. Our aim was to investigate the effects of PGE2 on pancreatic ductal adenocarcinoma cell growth and to characterize the underlying mechanisms. Methods: Human pancreatic ductal adenocarcinoma cell lines, Panc‐1 and MIA PaCa‐2, were treated with PGE2 in varying doses (0–10 &mgr;M). Effects on the phosphorylation of ERK1/2 were evaluated by Western blot. Colony formation was observed for cells treated with PGE2 for 11 days. DNA synthesis was determined by (3H)‐thymidine incorporation assay. Gene expression of E‐type prostaglandin (EP)2/EP4 receptors and their correlation with survival in patients with pancreatic ductal adenocarcinoma were assessed using the RNA‐Seq data set from The Cancer Genome Atlas Research Network. Results: PGE2 decreased the size and number of colonies in Panc‐1 but not MIA PaCa‐2 cells. In the Panc‐1 cells, PGE2 activated PKA/CREB and decreased phosphorylation of ERK1/2, which was reversed by an EP4 receptor antagonist, while an EP2 receptor antagonist had no effect. In contrast, in MIA PaCa‐2 cells, PGE2 had no effect on ERK1/2 phosphorylation. Treatment of both Panc‐1 and MIA PaCa‐2 cells with forskolin/IBMX decreased ERK1/2 phosphorylation. Finally, PGE2 decreased DNA synthesis only in Panc‐1 cells, which was reversed by an EP4 receptor antagonist. In human pancreatic ductal adenocarcinoma, high EP2 and low EP4 gene expression was correlated to worse median overall survival (15.6 vs 20.8 months, log‐rank P = .017). Conclusion: Our study provides evidence that PGE2 can inhibit directly pancreatic ductal adenocarcinoma cell growth through an EP4‐mediated mechanism. Together with our gene expression and survival analysis, this observation suggests a protective role of EP4 receptors in human pancreatic ductal adenocarcinoma that expresses E‐type prostaglandin receptors.

Abstract 5291: Signaling cross-talks in obesity-associated pancreatic cancer: Interaction between prostaglandin E2 signaling and mTOR pathway

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Obesity is a known risk factor for human cancers, including pancreatic cancer, and is associated with inflammation and insulin-resistance. The pro-inflammatory eicosanoid prostaglandin E2 (PGE2), which signals via EP receptors and cAMP, and insulin-like growth factor 1 (IGF-1), which is elevated in insulin resistance and activates the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling module, have both been shown to play critical roles in pancreatic cancer development and growth. The aim of this study was to investigate an unexplored crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In our studies, human pancreatic cancer cell lines were used, and the activation of signaling molecules was mainly assessed by Western blotting analysis. In multiple cell lines (Capan-2, HPAF-II, Mia PaCa-2, and PANC-1), PGE2 exposure increased intracellular cAMP levels measured by enzyme immunoassay, indicating the activation of Gs alpha-coupled EP receptors (EP2 and/or EP4). In PANC-1 cells, which showed the greatest responsiveness of cAMP induction, PGE2 dose- and time-dependently increased the phosphorylation of S6 ribosomal protein (S6rpSer235/236) that is downstream of mTORC1, suggesting a crosstalk between PGE2/cAMP and mTORC1. In addition, the effect of PGE2 on phospho-S6rp was mimicked by Forskolin, a pharmacological cAMP stimulator. Importantly, PGE2 enhanced the effect of IGF-1 on S6rp phosphorylation, supporting the existence of a positive reinforcement by the interaction between the two pathways. Interestingly, PGE2 and forskolin had no effect on Akt phosphorylation (Thr308 and Ser473), suggesting a link downstream of Akt. The activation of the mTORC-1 pathway upon PGE2 treatment paralleled an increase in the phosphorylation of cAMP response element-binding protein (CREB), a substrate of protein kinase A (PKA). In the presence of the PKA inhibitor H89, baseline phosphorylation of S6rp and PGE2-activated S6rp phosphorylation were reduced, indicating a role of PKA in the crosstalk. In summary, our data provide the first evidence of a crosstalk between the PGE2/EP2/cAMP and IGF-1/Akt/mTORC1 signaling pathways. Since both pathways are over-activated in obesity-associated cancers, this interaction may be of great importance in elucidating the tumor-promoting effects of obesity in pancreatic cancer. Ultimately, a detailed understanding of these molecular links may provide mechanistic targets for novel efficacious interventions devoid of adverse effects for pancreatic cancer in an increasingly obese population. Citation Format: Hui-Hua Chang, Guido Eibl. Signaling cross-talks in obesity-associated pancreatic cancer: Interaction between prostaglandin E2 signaling and mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5291. doi:10.1158/1538-7445.AM2014-5291

Clinical Advantages of Neoadjuvant Docetaxel (T) and Carboplatin (C) ± Trastuzumab (H) in Locally Advanced Breast Cancer (LABC).

Background : Neoadjuvant systemic treatment has become a standard choice for locally advanced breast cancer with many proposed benefits in achieving clear margins, preserving breasts, reducing relapse and improving survival. Recently, we completed a neoadjuvant trial studying the effects of 4 cycles of docetaxel (75 mg/m 2 ) and carboplatin (AUC=6) with or without trastuzumab (4mg/kg loading dose and 2 mg/kg weekly dose) on LABC. In this report, we assessed whether clinical complete response (cCR) or pathologic complete response (pCR) correlated better with having breast conservation surgery, fewer relapses and improved survival outcomes. Materials and Methods : Seventy-one of the 74 consented patients with T2-T4 non-metastatic breast cancer completed the preoperative treatment and had evaluable data on tumor response, surgical treatment and clinical outcome. We used the Kaplan Meier method to estimate survival probabilities and log rank test to compare relapse-free and survival curves. Results: Clinical outcomes from a preplanned 2-year analysis of a phase II neoadjuvant are reported. Of the 19 patients with pCR, 16 occurred in patients with cCR (n=32) and 3 were in the group of non-cCR (n=39). Although cCR overestimated tumor response, 84.2% pCR occurred in the cCR group and only 15.8% in the non-cCR group. The relapse-free survival at 2 and 3 years for pCR vs. non-pCR were 93.8% and 83.3% vs. 78.4% (p=0.122) and 58%, respectively; and for cCR vs. non-cCR were 80.9% and 65% vs. 83.9% and 64.3% (p=0.999). The pCR was also more predictive than cCR for overall survival. Of the 30 HER2 positive breast cancer, 15 received trastuzumab throughout the neoadjuvant and adjuvant phases for a total of 52 weeks. The remaining 15 patients received identical chemotherapy but trastuzumab was started after the surgery and continued for 52 weeks. While distinctively different pCR between the two treatment groups was expected, the better survival rate observed in the group receiving neoadjuvant TCH was not expected. Our study showed that pCR was strongly associated with a more frequent use of lumpectomy than the non-pCR group 63.2% vs. 36.5% (p=0.045). Conclusion : The effects of preoperative systemic treatment on LABC can be assessed both clinically and pathologically. Our data suggests that pathologic complete response was a better predictor for having breast conservation surgery and relapse-free survival rates. Our data also suggests that patients with HER2 positive breast cancer may benefit from receiving preoperative trastuzumab and chemotherapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1100.