Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion. Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking, suggesting compensation by other NHE family members. Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.

The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA ؉ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA-and TFSS-dependent mechanism, because strains 60190⌬cagA or 60190⌬cagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillin Tyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillin Tyr-118 were activated by 60190⌬cagA but not 60190⌬cagE, demonstrating CagA-independent signaling critical for cell motility. A ␤ 1 integrin-blocking antibody significantly inhibited JNK and paxillin Tyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through ␤ 1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the ␤ 1 integrin pathway.Helicobacter pylori infects one-half of the world's population, establishing a chronic infection in the gastric mucosa that persists for the lifetime of the host (1, 2). Although most infections only manifest as superficial gastritis, many progress to mucosal necrosis, ulceration, and atrophic gastritis, a precursor lesion of gastric adenocarcinoma (3). In animal studies, 37% of gerbils infected with virulent strains of H. pylori developed stomach tumors, demonstrating a direct link between H. pylori and gastric carcinogenesis (4). Additionally, epidemiological studies suggest that H. pylori infection increases the risk of developing gastric cancer 6-fold, emphasizing the importance of this bacterium in gastric carcinogenesis (5).H. pylori pathogenesis varies based on the expression of virulence factors used for bacterial colonization and disease progression. The vacA gene is encoded by virtually all H. pylori strains, but the intense vacuolation caused by VacA varies based on genetic mosaicism (6). Peptic ulceration strongly correlates with strains encoding the most active forms of VacA (6).The cag pathogenicity island (cag PAI) 3 contains 32 genes, many of which encode com...

Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.

Purpose Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases, RalA and RalB, are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole body irradiation but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR. Methods and Materials RalA, RalB and their major effectors RalBP1 and Sec5 were knocked down by stable expression of shRNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, γH2AX expression for double-strand DNA breaks (DSBs) and PARP cleavage for apoptosis. Results Knockdown of K-Ras, RalA or RalB reduced colony-forming ability post-IR and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR. Conclusions Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.

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Background:The hepatocyte growth factor (HGF) receptor, c-Met, is strongly implicated in late-stage cancer progression and poor patient prognosis. The stomach pathogen, Helicobacter pylori (H. pylori), was recently proposed to stimulate c-Met phosphorylation dependent upon interaction of c-Met with the bacterial CagA protein required for H. pylori-induced cancer cell motility and invasion.Materials and Methods:In this report, we employed short hairpin RNA (shRNA), western blot analysis using antibodies recognizing phosphorylation at discrete c-Met residues, and immunofluorescence microscopy to investigate the CagA-c-Met interaction.Results:The data showed that shRNA-mediated c-Met knockdown did not reduce H. pylori-induced cell motility, suggesting that c-Met was not required for motility. Surprisingly, c-Met knockdown did not reduce the level of an H. pylori-induced protein recognized by a phospho-c-Met antibody. This 125 kD protein was 10 kD smaller than c-Met, suggesting that H. pylori did not phosphorylate c-Met but cross-reacted with another protein. This hypothesis was confirmed when c-Met phosphorylation inhibitors did not lower the levels of the bacteria-induced 125 kD protein, and c-Met immunoprecipitation (IP) did not detect this 125 kD protein from H. pylori-treated lysates. This protein was identified as a product of antibody cross reactivity with phosphorylated CagA. We also confirmed that CagA interacts with c-Met, but this interaction may have caused previous authors to misinterpret phosphorylated CagA as c-Met phosphorylation. Finally, pretreatment with the proteasomal inhibitor, lactacystin, caused prolonged HGF-induced c-Met phosphorylation and facilitated a CagA-negative H. pylori to stimulate AGS cell motility, suggesting that sustained c-Met phosphorylation compensates for the loss of CagA-dependent signaling.Conclusions:These data demonstrate that H. pylori stimulates cancer cell motility independent of the c-Met receptor. We further hypothesize that although H. pylori does not target c-Met, the bacteria may still utilize c-Met effector signaling to stimulate CagA-independent cancer cell motility, which may provide a further mechanism of H. pylori-dependent gastric cancer progression.

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