EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

Evidence for the role of agonist binding frequency in receptor -mediated activation of adenylate cyclase

$\beta$-adrenergic receptor-mediated activation of adenylate cyclase exhibits an agonist-specific separation between the dose/response curve (characterized by the EC$\sb{50}$) and the dose/binding curve (characterized by the K$\sb{\rm d}$). Cyclase activity can be near-maximal when receptor occupancy is quite low (EC$\sb{50}$ $\ll$ K$\sb{\rm d}$). This separation between the binding and response curves can be explained by the assumption that the rate of cyclase activation is proportional to the concentration of agonist-bound receptors, since the receptor is mobile and can activate more than one cyclase (the Collision Coupling Model of Tolkovsky and Levitzki). Here it is established that agonist binding frequency plays an additional role in adenylate cyclase activation in S49 murine lymphoma cells. Using epinephrine (EC$\sb{50}$ = 10 nM, K$\sb{\rm d}$ = 2 $\mu$M), the rate of cyclase activation decreased by 80% when a small (1.5%) receptor occupancy was restricted (by addition of the antagonist propranolol) to a small number (1.5%) of receptors rather than being proportionally distributed among the cell's entire population of receptors. Thus adenylate cyclase activity is not proportional to receptor occupancy in all circumstances. Collisions between receptor and cyclase pairs apparently occur a number of times in rapid sequence (an encounter); the high binding frequency of epinephrine ensures that discontiguous regions of the cell surface experience some period of agonist-bound receptor activity per small unit time minimizing "wasted" collisions between activated cyclase and bound receptor within an encounter. A contribution of agonist binding frequency to activation is thus possible when: (1) the mean lifetime of the agonist-receptor complex is shorter than the mean encounter time, and (2) the absolute efficiency (intrinsic ability to promote cyclase activation per collision) of the agonist-receptor complex is high. These conclusions are supported by experiments using agonists of different efficiencies and binding frequencies. These results are formalized in the Encounter Coupling Model of adenylate cyclase activation, which takes into explicit account the agonist binding frequency, agonist affinity for the $\beta$-adrenergic receptor, agonist efficiency, encounter frequency and the encounter time between receptor and cyclase. ^