The mechanisms which allow cancer cells to adapt to the typical tumor microenvironment of low oxygen and glucose and high lactate are not well understood. GPR81 is a lactate receptor recently identified in adipose and muscle cells that has not been investigated in cancer. In the current study, we examined GPR81 expression and function in cancer cells. We found that GPR81 was present in colon, breast, lung, hepatocellular, salivary gland, cervical and pancreatic carcinoma cell lines. Examination of tumors resected from pancreatic cancer patients indicated that 94% (148/158) expressed high levels of GPR81. Functionally, we observed that the reduction of GPR81 levels using shRNA mediated silencing had little effect on pancreatic cancer cells cultured in high glucose, but led to the rapid death of cancer cells cultured in conditions of low glucose supplemented with lactate. We also observed that lactate addition to culture media induced the expression of genes involved in lactate metabolism including monocarboxylase transporters in control, but not in GPR81 silenced cells. In vivo, GPR81 expression levels correlated with the rate of pancreatic cancer tumor growth and metastasis. Cells in which GPR81 was silenced showed a dramatic decrease in growth and metastasis. Implantation of cancer cells in vivo was also observed to lead to greatly elevate levels of GPR81. These data support that GPR81 is important for cancer cell regulation of lactate transport mechanisms. Furthermore, lactate transport is important for the survival of cancer cells in the tumor microenvironment.

Background & Aims Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development, and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity, and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice. Methods We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1 -Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated ERK, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs. Results Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs. Conclusions In mice, a HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis, and development of PanINs and PDAC. This mechanism could be involved in the association between risk for PDAC and HFDs.

Pancreatic stellate cells (PSCs) have been recognized as the principal cells responsible for the production of fibrosis in PDAC. Recently PSCs have been noted to share characteristics with cells of monocyte-macrophage lineage (MML cells). Thus, we tested whether PSCs could be targeted with the nitrogen-containing bisphosphonates (NBPs) [pamidronate (Pam) or zoledronic acid (ZA)], which are potent MML cell inhibitors. In addition, we tested NBPs treatment combination with nanoparticle albumin-bound paclitaxel (nab-paclitaxel) to enhance antitumor activity. In vitro we observed that PSCs possess α-naphthyl butyrate esterase (ANBE) enzyme activity, a specific marker of MML cells. Moreover NBPs inhibited PSCs proliferation, activation, release of macrophage chemoattractant protein-1 (MCP-1) and type I collagen expression. NBPs also induced PSC apoptosis and cell cycle arrest in the G1 phase. In vivo, NBPs inactivated PSCs; reduced fibrosis; inhibited tumor volume, tumor weight, peritoneal dissemination, angiogenesis, and cell proliferation; and increased apoptosis in an orthotopic murine model of PDAC. These in vivo antitumor effects were enhanced when NBPs were combined with nab-paclitaxel but not gemcitabine (Gem). Our study suggests that targeting PSCs and tumor cells with NBPs in combination with nab-paclitaxel may be a novel therapeutic approach to PDAC.

<p>PDF file - 163K, Supplementary Figure 1: Lentiviral shRNA mediated knockdown or ectopic expression of GPR81. Supplementary Figure 2: Lentiviral shRNA mediated knockdown of GPR81 is associated with reduced levels of MCT1. Supplemental Table 1: Primer sequences for quantitative RT-PCR.</p>

<p>PDF file - 163K, Supplementary Figure 1: Lentiviral shRNA mediated knockdown or ectopic expression of GPR81. Supplementary Figure 2: Lentiviral shRNA mediated knockdown of GPR81 is associated with reduced levels of MCT1. Supplemental Table 1: Primer sequences for quantitative RT-PCR.</p>