Lyn, a Src family kinase, regulates activation of epidermal growth factor receptors in lung adenocarcinoma cells.

BACKGROUND: Activation of receptors for growth factors on lung epithelial cells is essential for transformation into tumor cells, supporting their viability and proliferation. In most lung cancer patients, EGFR is constitutively activated without evidence of mutation. Defining mechanisms for constitutive activation of EGFR could elucidate additional targets for therapy of lung cancers. METHODS: The approach was to identify lung cancer cell lines with constitutively activated EGFR and use systematic selection of inhibitors to evaluate their effects on specific EGFR phosphorylations and downstream signaling pathways. Interactions between receptors, kinases, and scaffolding proteins were investigated by co-immunoprecipitation plus Western blotting. RESULTS: The results revealed a dependence on Src family of tyrosine kinases for downstream signaling and cell growth. Lyn, a Src family kinase functional in normal and malignant B-lymphocytes, was a defining signal transducer required for EGFR signaling in Calu3 cell line. Src family kinase activation in turn, was dependent on PKCßII. Lyn and PKC exist in membrane complexes of RACK1 and in association with EGFR which pairs with other receptor partners. Silencing of Lyn expression with interfering siRNA decreased EGFR activation and cell viability. CONCLUSIONS: The importance of Src family kinases and PKCßII in the initiation of the EGFR signaling pathway in lung tumor cells was demonstrated. We conclude that phosphorylation of EGFR is mediated through PKCßII regulation of Lyn activation, and occurs in association with RACK1 and Cbp/PAG proteins. We suggest that protein complexes in cell membranes, including lipid rafts, may serve as novel targets for combination therapies with EGFR and Src Family Kinase inhibitors in lung cancer.

Major histocompatibility complex class I-related chain A/B (MICA/B) expression in tumor tissue and serum of pancreatic cancer: role of uric acid accumulation in gemcitabine-induced MICA/B expression.

BACKGROUND: Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind the immunoreceptor NKG2D and play an important role in mediating the cyotoxicity of NK and T cells. In this study, we sought to study MICA/B expression in pancreatic cancer and to determine whether and how genotoxic drugs such as gemcitabine can affect MICA/B expression and natural killer cytotoxity. METHODS: Seven pancreatic cancer cell lines were analyzed for MICA/B expression by flow cytometry and for their sensitivity to NK-92 cell killing by a 51Cr release assay. MICA/B expression in tumor tissues and sera of pancreatic cancer was analyzed by immunohistochemical staining (IHC) and ELISA, respectively. RESULTS: Two MICA/B-positive cell lines were sensitive to the cytotoxic activity of NK-92 cells. Other two MICA/B-positive cell lines and three MICA/B-negative cell lines were resistant to NK-92 cell killing. MICA/B expression was positive in 17 of 25 (68%) pancreatic ductal adenocarcinomas but not in normal pancreatic ductal epithelial cells. Serum MICA/B levels were significantly elevated in patients with pancreatic adenocarcinomas but did not correlate with the stage of pancreatic cancer and patient survival. Gemcitabine therapy led to increased serum MICA levels in 6 of 10 patients with detectable serum MICA. Allopurinol, an inhibitor of xanthine oxidoreductase that converts xanthine to uric acid, blocked uric acid production, MICA/B expression, and sensitivity to NK-92 cell killing toward a PANC-1 cancer cell line exposed to radiation and two genotoxic drugs, gemcitabine and 5-fluorouracil. CONCLUSIONS: The levels of MICA/B expression in serum and tissue of pancreatic cancer are elevated. DNA damage-induced MICA/B expression is mediated through increased uric acid production.

Current immunotherapeutic strategies in pancreatic cancer.

The immune systems of patients with newly diagnosed pancreatic cancers are functional, with T-cell responses capable of responding to tumor antigen presentation. Pancreatic tumors have been demonstrated to express tumor antigens as mutated, altered, underglycosylated and/or inappropriately overexpressed proteins. Considering these two facts, it should be possible for patients' bodies to recognize their tumors as foreign and to reject them. A number of clinical trials have been initiated to exploit this immune activation to eradicate or stabilize tumor growth. Immunotherapeutic trials include the specific testing of a variety of tumor vaccines, of cytokines as adjuvants or directed cytotoxicity, and of monoclonal antibodies to target specific molecules. This article reviews evidence for immune-cell activation and function in patients with pancreatic cancer, and evidence that pancreatic tumor cells express tumor antigens, or mutated (or altered) proteins. Nevertheless, tumors survive immune attacks by producing products that help them to circumvent effector T cells. The article thus examines complications of immune evasion by cancer cells, as well as the challenges of trying to exploit the immune system in solid tumors where tumor cell products can turn off invading immune T cells set to kill them. Finally, the article discusses the choices of a variety of clinical trials using immune modulation for patients with pancreatic cancer.

Clinicopathological significance of major histocompatibility complex class I-related chain a and B expression in thyroid cancer.

CONTEXT: Major histocompatibility complex class I-related chains A and B (MICA/B) are two stress-inducible ligands for the immunoreceptor NKG2D that is expressed on cytotoxic T cells and natural killer (NK) cells. It is not known whether MICA/B expression is up-regulated in thyroid cancer as a result of oncogene activation. OBJECTIVE: The objective of the investigation was to study MICA/B expression and regulation in thyroid cancer and its role in mediating the cytotoxicity of NK cells. METHODS: MICA/B expression in thyroid cancer was analyzed by immunohistochemical staining. Cell surface MICA/B levels in thyroid tumor cell lines and fresh tumor cells were analyzed by flow cytometric analysis. The susceptibility of thyroid tumor cells to NK cell killing was tested by using (51)Cr release assay. RESULTS: MICA/B was expressed at moderate or high levels in 18 of 39 papillary thyroid carcinomas and four of eight anaplastic thyroid carcinomas. MICA/B expression was confirmed by flow cytometric analysis in three fresh thyroid neoplasms. MICA/B expression was detected in eight of 10 thyroid tumor cell lines and correlated with their sensitivity to killing by the NKG2/D-positive NK-92 cells. Blocking of NKG2D and MICA/B interaction by specific antibodies partially led to the inhibition of NK-92 cell-mediated cytotoxicity. The MAPK inhibitors were able to block MICA/B expression in MRO87 and HeLa cells. Transient transfection of mutant BRAF and RAS oncogenes led to increased MICA/B expression in 293 cells and WRO82 cells. CONCLUSION: MICA/B expression is up-regulated in thyroid cancer, probably due to the activation of the MAPK pathway. MICA/B in thyroid cancer plays an important role in NK-92 cell-mediated cytotoxicity.

PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells by preventing caspase 8 activation.

Studies to investigate signal transduction pathways that support viability and prevent apoptosis of chronic lymphocytic leukemia cells (CLL) were initiated as a result of microarray cDNA analyses which revealed expression of genes whose products regulate cell cycle progression. Immunoblots revealed translation of several genes including caspases, cyclin D1, and the PI3-kinase dependent, survival kinase, Akt. Akt was found to be activated. Inhibition of PI3-kinase with specific inhibitor, LY294002, led to the induction of apoptosis that was caspase 8 dependent, but independent of Akt as LY294002 did not depress a high basal level of Akt activity found in CLL cells. Phosphorylation of Akt was maintained, enzymatic activity undiminished, and phosphorylation of substrates sustained. Caspases, however were activated, PARP cleaved and DNA fragmented. Caspase inhibitors revealed that initiator caspase 8 was required for classic apoptosis when PI3-kinase was inhibited, and specific activity assays demonstrated its early activation. GSK-3beta a kinase regulated via PI3-kinase dependent, down-stream kinases, was responsible for regulating cyclin D1 levels in CLL cells, but neither GSK-3beta nor calpain was responsible for induction of apoptosis, or activation of executioner caspase 3, following LY294002 treatment. PI3-kinase mediated protection against caspase activation in CLL B-cells therefore is not mediated through classic Akt survival pathways. The data further support the hypothesis that signal transducing, membrane associated receptors triggered by extrinsic factors, maintain CLL leukemic B-cell survival in vivo by preventing caspase activation.

Advances in therapeutic vaccines for pancreatic cancer.

Pancreatic cancer is one of the most difficult-to-treat cancers. Despite surgical resection, radiation and/or chemotherapy, greater than 94% of people with pancreatic cancer do not survive beyond 5 years. In fact, median survival after diagnosis of metastatic pancreatic cancer is 4.5 months. The majority of patients are diagnosed with nonresectable, metastatic disease, and chemotherapy only extends their median survival by less than 2 months with only 18% of those treated surviving beyond 1 year. Despite the severity of their disease, most patients exhibit tumor specific cellular immunity to their pancreatic cancer antigens. Obviously their immunity is ineffective in preventing tumor growth. Recent studies have demonstrated that the tumor microenvironment may hold the key to determining the nature of the tumors' ability to escape from immune attack. Preliminary clinical trials have suggested that blocking these escape mechanisms may result in survival benefit to the patients, and phase I and II clinical trials with tumor vaccines have led to some survival benefits. Perhaps combining therapies directed against immune escape mechanisms with tumor vaccines will result in even greater survival benefit for patients with pancreatic cancer. While therapeutic vaccines for pancreatic cancers have been reviewed previously (Plate, 2011), updates on recent preliminary reports of two clinical vaccine trials are worthy of our attention.

Effect of pemetrexed on innate immune killer cells and adaptive immune T cells in subjects with adenocarcinoma of the pancreas.

Baseline levels of innate and adaptive immune cell functions were studied in patients with pancreatic cancer. The effects of pemetrexed were measured at 7 and 14 days after initial therapy then 14 days after combination therapy with gemcitabine. Pretherapy levels of absolute numbers of natural killer (NK) cells positively correlated with survival. Cytolytic units of NK activity correlated positively with NK cell numbers. Pemetrexed decreased NK cytolytic units to significance when combined with gemcitabine. Pemetrexed increased intracellular accumulation of interferon gamma (IFNγ) in NK cells that correlated negatively with survival. Addition of gemcitabine decreased IFNγ-producing NK cells to baseline. Memory (CD45RO*) T cells enumerated at baseline correlated negatively with survival but were decreased by pemetrexed therapy. Memory T cells were increased in subjects with greater B7-H3 expression in tumor tissue, whereas OX40*-activated total T cells and helper T-cell subset were decreased. FoxP3*, CD8* T cells correlated positively with progression-free interval and survival. In conclusion, innate NK-cell immunity and FoxP3*, CD8* T cells seemed beneficial to pancreatic cancer patients. Higher levels of B7-H3 expression in pancreatic tumors were detrimental to effective immunity. Although pemetrexed therapy increased activation of a subset of NK cells to produce IFNγ, addition of gemcitabine abated those responses, decreasing IFNγ-producing NK cells, whereas NK cells producing interleukin-2 without IFNγ at this timepoint positively correlated with survival. Innate immunity and adaptive immunity thus are important in defense against pancreatic cancer. Progression-free interval and survival were longer than observed in a phase III trial where gemcitabine preceded pemetrexed suggesting that a larger trial of pemetrexed preceding gemcitabine is warranted.

Clinical trials of vaccines for immunotherapy in pancreatic cancer.

A greater understanding of the molecular events that trigger oncogenesis and events that negatively regulate immune responses has allowed for the development of targeted therapies with specific vaccines to match tumor antigens coupled with immunotherapy specifically directed against that tumor's immunosuppressive microenvironment. In order to be effective, vaccine therapies need to both expand the immune response to tumors and overcome immunosuppressive microenvironments therein. Specifically, targeted therapy must be personalized for each cancer patient. While the idea of personalized targeted therapy may seem like a daunting task, it may not be that difficult as it could involve a relatively simple genetic test to identify gene mutations and additional immunohistochemical staining of tumors with antibodies directed against markers of negative immune regulation. The additional cost to personalize cancer therapy with these diagnostic tests is relatively small in comparison to the cost afforded to our healthcare system when inappropriate targeting therapies are administered to patients whose tumors do not express the targets of either the vaccine or the immune modulator. Despite the large cost, cancer patients whose tumors lack the targets of these therapies often receive no benefit from the therapy. The most illogical approach is to develop a study design and perform clinical trials of potential novel targeting drugs without knowledge or confirmation that the patients' tumors express the targets. Current cancer trials for pancreatic cancer patients are discussed in this article.

Countering tumor-induced immunosuppression during immunotherapy for pancreatic cancer.

BACKGROUND: Vaccines for pancreatic cancer have been challenged by a number of factors, especially the immunosuppressive microenvironment within the tumor that allows for escape from immune surveillance. OBJECTIVE/METHODS: We sought to identify results that define mechanisms of pancreatic-cancer-associated immunosuppression and strategies that might be useful to overcome them thereby resulting in effective immune responses to cancer vaccines capable of deleting pancreatic cancer cells. RESULTS/CONCLUSION: Immunosuppressive tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg) reside in tumors, and their products along with tumor derived products (such as VEGF, TGFbeta and IL-10), create a microenvironment that counters immune activation and attack. Immunotherapy with cancer vaccines must include strategies to modulate these immunosuppressive cell types and tumor byproducts. Clinical trials are beginning to test these strategies.

Elevated serum heparanase-1 levels in patients with pancreatic carcinoma are associated with poor survival.

BACKGROUND: It has previously been shown that heparanase-1 (HPR1), an endoglycosidase, is up-regulated in pancreatic carcinoma. The purpose of this study was to test whether serum HPR1 levels in pancreatic carcinoma patients are elevated, and whether higher serum HPR1 levels are associated with a shortened survival. METHODS: Serum HPR1 levels in 40 healthy donors, 31 pancreatic carcinoma patients, and 11 patients treated with gemcitabine were measured by a novel enzyme-linked immunoadsorbent assay. HPR1 expression in tumors was analyzed by immunohistochemical staining. Patient overall survival time was determined according to the Kaplan-Meier method, and their difference was evaluated by the log-rank test. A P value<0.05 was considered statistically significant. RESULTS: The mean serum HPR1 activity in pancreatic carcinoma patients was 439+/-14 units/mL, compared with 190+/-4 units/mL in the control serum samples from healthy donors. Serum HPR1 levels were significantly higher in patients with HPR1-positive tumors (660+/-62 units/mL) compared with those with HPR1-negative tumors (241+/-14 units/mL). The mean survival of 19 pancreatic carcinoma patients with serum HPR1 activity>300 units/mL was 7.9+/-0.2 months, whereas the mean survival of 12 patients with serum HPR1 activity<300 units/mL was 13.3+/-0.6 months. A Kaplan-Meier plot of the patient survival curve followed by log-rank test revealed that patients in the high serum HPR1 group had a significantly shorter survival compared with those in the low serum HPR1 group. Mean serum HPR1 activity decreased by 64% in 11 pancreatic carcinoma patients after 2 weeks of treatment with gemcitabine. CONCLUSIONS: Serum HPR1 activity in pancreatic carcinoma patients was found to be significantly elevated, in particular in those with HPR1-positive tumors. Increased serum HPR1 activity was associated with a shorter survival in patients with pancreatic carcinoma patients.

Immunobiotherapy directed against mutated and aberrantly expressed gene products in pancreas cancer.

Genetic alterations are responsible for the development of cancer in ductal cells of the pancreas. These genetic changes result in abnormal molecular expression of proteins that are involved in cell proliferation, cell cycle control and adhesion. Some of the genetic mutations result in aberrant proteins that can be recognized as novel or foreign by cells of innate and adaptive immune systems. These are appropriate targets for therapeutic intervention which may involve immunobiologic approaches. These approaches may be less effective because of immune escape mechanisms developed by tumor cells within the microenvironment of the tumor mass. Immunobiotherapy intervention of pancreas cancer must circumvent these obstacles and integrate effective immunotherapy with molecularly targeted approaches to pancreas cancer intervention.

Effect of gemcitabine on immune cells in subjects with adenocarcinoma of the pancreas.

Effects of gemcitabine (Gemzar) on immune cells were examined in pancreas cancer patients to determine whether it was immunosuppressive, or potentially could be combined with vaccines or other immunotherapy to enhance patient's responses to their tumors. Blood was obtained at five time-points, before therapy, 3-4 days after initial gemcitabine infusion and immediately preceding three additional weekly infusions. Effects on T-cell subsets, B-cells, myeloid dendritic cell precursors, antigen presenting cells (APC), activated/memory, and naive cells were examined. Functional activity was measured by intracellular staining for cytokines before and after T-cell activation, and by interferon gamma production in EliSpot responses to tumor presentation. Although absolute lymphocyte counts decreased with the initial treatment with gemcitabine infusion, the counts stabilized during subsequent treatments, then returned within normal ranges seven days after the fourth treatment so that the absolute lymphocyte count no longer differed significantly from that prior to treatment. These effects on absolute lymphocyte counts were mirrored by statistically significant decreases in absolute numbers of CD3 and CD20 lymphocytes during these time periods. The proportions of T and B-cells, however did not change significantly with therapy, although significance changes were observed in some specialized subsets. A decrease in the proportions of the major BDCA-1+, CD1b myeloid dendritic cell subset and a reciprocal increase in the minor BDCA-3+ dendritic cell subsets resulted at 3-4 days, then their levels returned to normal. No significant changes in percentages of CD86 and CD80 APCs or CD4+, CD25+ T-cells were documented. Increased percentages of CD3+, CD45RO+ memory lymphocytes reached significance at day 7, then declined to statistically significant decrease at days 14 and 21 after the second and third infusions, respectively. Immune T-cells were functional in pancreas cancer patients treated with gemcitabine. The data suggest that gemcitabine therapy may decrease memory T-cells and promote naive T-cell activation. We conclude that gemcitabine therapy (1) is not immunosuppressive and (2) may enhance responses to specific vaccines or immunotherapy administered to activate or support immune responses directed toward driving effector immunity to cancer cells.