Complex interactions of lovastatin with 10 chemotherapeutic drugs: a rigorous evaluation of synergism and antagonism.

BACKGROUND: Evidence bearing on the role of statins in the prevention and treatment of cancer is confounded by the diversity of statins, chemotherapeutic agents and cancer types included in the numerous published studies; consequently, the adjunctive value of statins with chemotherapy remains uncertain. METHODS: We assayed lovastatin in combination with each of ten commonly prescribed chemotherapy drugs in highly reproducible in vitro assays, using a neutral cellular substrate, Saccharomyces cerevisiae. Cell density (OD600) data were analyzed for synergism and antagonism using the Loewe additivity model implemented with the Combenefit software. RESULTS: Four of the ten chemotherapy drugs - tamoxifen, doxorubicin, methotrexate and rapamycin - exhibited net synergism with lovastatin. The remaining six agents (5-fluorouracil, gemcitabine, epothilone, cisplatin, cyclophosphamide and etoposide) compiled neutral or antagonistic scores. Distinctive patterns of synergism and antagonism, often coexisting within the same concentration space, were documented with the various combinations, including those with net synergism scores. Two drug pairs, lovastatin combined with tamoxifen or cisplatin, were also assayed in human cell lines as proof of principle. CONCLUSIONS: The synergistic interactions of tamoxifen, doxorubicin, methotrexate and rapamycin with lovastatin - because they suggest the possibility of clinical utility - merit further exploration and validation in cell lines and animal models. No less importantly, strong antagonistic interactions between certain agents and lovastatin argue for a cautious, data-driven approach before adding a statin to any chemotherapeutic regimen. We also urge awareness of adventitious statin usage by patients entering cancer treatment protocols.

Double-negative (CD27-IgD-) B cells are expanded in NSCLC and inversely correlate with affinity-matured B cell populations.

BACKGROUND: The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells differentiate into a variety of subsets with differing characteristics and functions. To date, there is limited information on the specific B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are CD79A+CD27-IgD-. These CD27-IgD- (double-negative) B cells have previously been characterized as unconventional memory B cells and have been detected in some autoimmune diseases and in the elderly population but have not been detected previously in tumor tissue. METHODS: A total of 15 fresh untreated NSCLC tumors and 15 matched adjacent lung control tissues were dissociated and analyzed by intracellular flow cytometry to detect the B cell-related markers CD79A, CD27 and IgD. All CD79A+ B cells subsets were classified as either naïve (CD27-IgD+), affinity-matured (CD27+IgD-), early memory/germinal center cells (CD27+IgD+) or double-negative B cells (CD27-IgD-). Association of double-negative B cells with clinical data including gender, age, smoking status, tumor diagnosis and pathologic differentiation status were also examined using the logistic regression analysis for age and student's t-test for all other variables. Associations with other B cell subpopulations were examined using Spearman's rank correlation. RESULTS: We observed that double-negative B cells were frequently abundant in lung tumors compared to normal adjacent controls (13 out of 15 cases), and in some cases made up a substantial proportion of the total B cell compartment. The presence of double-negative cells was also found to be inversely related to the presence of affinity-matured B cells within the tumor, Spearman's coefficient of - 0.76. CONCLUSIONS: This study is the first to observe the presence of CD27-IgD- double-negative B cells in human NSCLC and that this population is inversely correlated with traditional affinity-matured B cell populations.

Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.

Activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling and the consequent induction of transformation by overexpressed 14-3-3γ protein require specific amino acids within 14-3-3γ N-terminal variable region II.

Members of the 14-3-3 superfamily regulate numerous cellular functions by binding phosphoproteins. The seven human isoforms (and the myriad of other eukaryotic 14-3-3 proteins) are highly conserved in amino acid sequence and secondary structure, yet there is abundant evidence that the various isoforms manifest disparate as well as common functions. Several of the human 14-3-3 isoforms are dysregulated in certain cancers and thus have been implicated in oncogenesis; experimentally, 14-3-3γ behaves as an oncogene, whereas 14-3-3σ acts as a tumor suppressor. In this study, we sought to localize these opposing phenotypes to specific regions of the two isoforms and then to individual amino acids therein. Using a bioinformatics approach, six variable regions (VRI-VRVI) were identified. Using this information, two sets of constructs were created in which N-terminal portions (including either VRI-IV or only VRI and VRII) of 14-3-3γ and 14-3-3σ were swapped; NIH3T3 cells overexpressing the four chimeric proteins were tested for transformation activity (focus formation, growth in soft agar) and activation of PI3K and MAPK signaling. We found that the specific phenotypes of 14-3-3γ are associated with the N-terminal 40 amino acids (VRI and VRII); in like fashion, VRI and VRII of 14-3-3σ dictated its tumor suppressor function. Using individual amino acid substitutions within the 14-3-3γ VRII, we identified two residues required for and two contributing to the γ-specific phenotypes. Our observations suggest that isoform-specific phenotypes are dictated by a relatively few amino acids within variable regions.

Encapsulated Cells for the Treatment of Diabetes: Danger of Acute Hypoglycemia Following Injury?

Transplants comprised of encapsulated islets have shown promise in treating insulin-dependent diabetes. A question raised in the scientific and clinical communities is whether the insulin released from an implanted encapsulation device damaged in an accident could cause a serious hypoglycemic event. In this commentary, we consider the different types of damage that a device can sustain, including the encapsulation membrane and the islets within, and the amount of insulin released in each case. We conclude that the probability that device damage would cause an adverse hypoglycemic event is indeed very low.

Accelerated absorption of regular insulin administered via a vascularizing permeable microchamber implanted subcutaneously in diabetic Rattus norvegicus.

In Type 1 diabetes patients, even ultra-rapid acting insulins injected subcutaneously reach peak concentrations in 45 minutes or longer. The lag time between dosing and peak concentration, as well as intra- and inter-subject variability, render prandial glucose control and dose consistency difficult. We postulated that insulin absorption from subcutaneously implantable vascularizing microchambers would be significantly faster than conventional subcutaneous injection. Male athymic nude R. norvegicus rendered diabetic with streptozotocin were implanted with vascularizing microchambers (single chamber; 1.5 cm2 surface area per side; nominal volume, 22.5 µl). Plasma insulin was assayed after a single dose (1.5 U/kg) of diluted insulin human (Humulin®R U-100), injected subcutaneously or via microchamber. Microchambers were also implanted in additional animals and retrieved at intervals for histologic assessment of vascularity. Following conventional subcutaneous injection, the mean peak insulin concentration was 22.7 (SD 14.2) minutes. By contrast, when identical doses of insulin were injected via subcutaneous microchamber 28 days after implantation, the mean peak insulin time was shortened to 7.50 (SD 4.52) minutes. Peak insulin concentrations were similar by either route; however, inter-subject variability was reduced when insulin was administered via microchamber. Histologic examination of tissue surrounding microchambers showed mature vascularization on days 21 and 40 post-implantation. Implantable vascularizing microchambers of similar design may prove clinically useful for insulin dosing, either intermittently by needle, or continuously by pump including in "closed loop" systems, such as the artificial pancreas.

The Potential of Topoisomerase Inhibitor-Based Antibody-Drug Conjugates.

DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody-drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs.

P53 suppresses expression of the 14-3-3 gamma oncogene.

BACKGROUND: 14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3 gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro. METHODS: qRTPCR and Western blot analysis were performed to examine 14-3-3 gamma expression in non-small cell lung cancers (NSCLC). Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3 gamma promoter. RESULTS: Quantitative rtPCR results showed that the expression level of 14-3-3 gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3 gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3 gamma expression. CONCLUSION: Increased expression of 14-3-3 gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3 gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

Peptide Adjuvant to Invigorate Cytolytic Activity of NK Cells in an Obese Mouse Cancer Model.

Cancer patients who are overweight compared to those with normal body weight have obesity-associated alterations of natural killer (NK) cells, characterized by poor cytotoxicity, slow proliferation, and inadequate anti-cancer activity. Concomitantly, prohibitin overexpressed by cancer cells elevates glucose metabolism, rendering the tumor microenvironment (TME) more tumor-favorable, and leading to malfunction of immune cells present in the TME. These changes cause vicious cycles of tumor growth. Adoptive immunotherapy has emerged as a promising option for cancer patients; however, obesity-related alterations in the TME allow the tumor to bypass immune surveillance and to down-regulate the activity of adoptively transferred NK cells. We hypothesized that inhibiting the prohibitin signaling pathway in an obese model would reduce glucose metabolism of cancer cells, thereby changing the TME to a pro-immune microenvironment and restoring the cytolytic activity of NK cells. Priming tumor cells with an inhibitory the prohibitin-binding peptide (PBP) enhances cytokine secretion and augments the cytolytic activity of adoptively transferred NK cells. NK cells harvested from the PBP-primed tumors exhibit multiple markers associated with the effector function of active NK cells. Our findings suggest that PBP has the potential as an adjuvant to enhance the cytolytic activity of adoptively transferred NK cells in cancer patients with obesity.

Recruitment of Dual-Career Academic Medicine Couples.

Today it is not uncommon to discover that a candidate for a faculty position has a partner or spouse who is also an academician, adding complexity to the recruitment process. Here, the authors address two practical obstacles to the recruitment of faculty who have an academic partner: dual recruitment and conflict of interest. The authors have found that tandem recruitment works best when suitable positions for both spouses are first identified so that recruitment can proceed synchronously. This approach decreases misperceptions of favoritism toward either's candidacy. Managing conflict of interest, generated by the appointment of one spouse in a supervisory position over the other, requires a proactive, transparent, well-designed plan. After canvassing human resource policies and conducting interviews with national academic leaders, the authors have developed an administrative structure that places "key" decisions (hiring and retention; promotion and tenure; salary, bonuses, and benefits; performance evaluations; and disciplinary matters) regarding the supervised spouse in the jurisdiction of an alternative administrator or committee. The authors also offer suggestions both for mitigating misperceptions of bias in day-to-day decisions and for the support and mentoring of the supervised partner or spouse.

The induction of endoreduplication and polyploidy by elevated expression of 14-3-3γ.

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.

Using logistic regression to improve the prognostic value of microarray gene expression data sets: application to early-stage squamous cell carcinoma of the lung and triple negative breast carcinoma.

BACKGROUND: Numerous microarray-based prognostic gene expression signatures of primary neoplasms have been published but often with little concurrence between studies, thus limiting their clinical utility. We describe a methodology using logistic regression, which circumvents limitations of conventional Kaplan Meier analysis. We applied this approach to a thrice-analyzed and published squamous cell carcinoma (SQCC) of the lung data set, with the objective of identifying gene expressions predictive of early death versus long survival in early-stage disease. A similar analysis was applied to a data set of triple negative breast carcinoma cases, which present similar clinical challenges. METHODS: Important to our approach is the selection of homogenous patient groups for comparison. In the lung study, we selected two groups (including only stages I and II), equal in size, of earliest deaths and longest survivors. Genes varying at least four-fold were tested by logistic regression for accuracy of prediction (area under a ROC plot). The gene list was refined by applying two sliding-window analyses and by validations using a leave-one-out approach and model building with validation subsets. In the breast study, a similar logistic regression analysis was used after selecting appropriate cases for comparison. RESULTS: A total of 8594 variable genes were tested for accuracy in predicting earliest deaths versus longest survivors in SQCC. After applying the two sliding window and the leave-one-out analyses, 24 prognostic genes were identified; most of them were B-cell related. When the same data set of stage I and II cases was analyzed using a conventional Kaplan Meier (KM) approach, we identified fewer immune-related genes among the most statistically significant hits; when stage III cases were included, most of the prognostic genes were missed. Interestingly, logistic regression analysis of the breast cancer data set identified many immune-related genes predictive of clinical outcome. CONCLUSIONS: Stratification of cases based on clinical data, careful selection of two groups for comparison, and the application of logistic regression analysis substantially improved predictive accuracy in comparison to conventional KM approaches. B cell-related genes dominated the list of prognostic genes in early stage SQCC of the lung and triple negative breast cancer.

Toward maintaining the genome: DNA damage and replication checkpoints.

DNA checkpoints play a significant role in cancer pathology, perhaps most notably in maintaining genome stability. This review summarizes the genetic and molecular mechanisms of checkpoint activation in response to DNA damage. The major checkpoint proteins common to all eukaryotes are identified and discussed, together with how the checkpoint proteins interact to induce arrest within each cell cycle phase. Also discussed are the molecular signals that activate checkpoint responses, including single-strand DNA, double-strand breaks, and aberrant replication forks. We address the connection between checkpoint proteins and damage repair mechanisms, how cells recover from an arrest response, and additional roles that checkpoint proteins play in DNA metabolism. Finally, the connection between checkpoint gene mutation and genomic instability is considered.