Inorganic arsenic: A non-genotoxic carcinogen.

Inorganic arsenic induces a variety of toxicities including cancer. The mode of action for cancer and non-cancer effects involves the metabolic generation of trivalent arsenicals and their reaction with sulfhydryl groups within critical proteins in various cell types which leads to the biological response. In epithelial cells, the response is cell death with consequent regenerative proliferation. If this continues for a long period of time, it can result in an increased risk of cancer. Arsenicals do not react with DNA. There is evidence for indirect genotoxicity in various in vitro and in vivo systems, but these involve exposures at cytotoxic concentrations and are not the basis for cancer development. The resulting markers of genotoxicity could readily be due to the cytotoxicity rather than an effect on the DNA itself. Evidence for genotoxicity in humans has involved detection of chromosomal aberrations, sister chromatid exchanges in lymphocytes and micronucleus formation in lymphocytes, buccal mucosal cells, and exfoliated urothelial cells in the urine. Numerous difficulties have been identified in the interpretation of such results, including inadequate assessment of exposure to arsenic, measurement of micronuclei, and potential confounding factors such as tobacco exposure, folate deficiency, and others. Overall, the data strongly supports a non-linear dose response for the effects of inorganic arsenic. In various in vitro and in vivo models and in human epidemiology studies there appears to be a threshold for biological responses, including cancer.

KIBRA regulates aurora kinase activity and is required for precise chromosome alignment during mitosis.

The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser(83) by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser(539), probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.

Histone deacetylase inhibitor belinostat represses survivin expression through reactivation of transforming growth factor beta (TGFbeta) receptor II leading to cancer cell death.

Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor ß receptor II (TGFßRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFß signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFß signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFß/protein kinase A signaling pathway. Induction of TGFßRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFßRII might potentially benefit from the use of this histone deacetylase inhibitor.

Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles.

Systemic AAV (adeno-associated virus) gene therapy is a promising approach for the treatment of inborn errors of metabolism, but questions remain regarding its potency and durability. Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been shown to block the formation of neutralizing anti-capsid antibodies, thereby enabling vector re-administration. Here, we further demonstrate that ImmTOR admixed with AAV vectors also enhances hepatic transgene expression at the initial dose of AAV vector, independent of its effects on adaptive immunity. ImmTOR enhances AAV trafficking to the liver, resulting in increased hepatic vector copy numbers and transgene mRNA expression. Enhanced transgene expression occurs through a mechanism independent of the AAV receptor and cannot be replicated in vivo with free rapamycin or empty nanoparticles. The multipronged mechanism of ImmTOR action makes it an attractive candidate to enable more efficient transgene expression at first dose while simultaneously inhibiting adaptive responses against AAV to enable repeat dosing.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Effect of polyhexamethylene biguanide on rat liver.

Polyhexamethylene biguanide (PHMB), an amphiphilic polymeric biocide, increased liver tumor incidence in male and female rats at 1000 and 1500 mg/L in drinking water, but not at 500 mg/L in previous studies. In another study, PHMB administered in diet at 4000 mg/kg was negative for hepatocellular tumors. The present studies evaluated bioavailability and distribution of PHMB administered in drinking water and diet and possible modes of action (MOA). PHMB in drinking water was unpalatable during the first 3 days, resulting in markedly decreased food consumption and decreased body weight. Ki-67 labeling index was increased in hepatocytes and endothelial cells dose responsively with PHMB administered in drinking water but not diet. Vitamin E had no effect on this. There was no cytotoxicity by histopathology or serum enzymes, and no increase in cytokines TNFα, IL-1α or NF-κB. Focal iron deposition in sinusoidal lining cells was detected. Microarray analyses were non-contributory. No effect on CAR or PPARα activation was detected. 14C-PHMB administered at 500, 1000, or 1500 mg/L in the drinking water or 4000 mg/kg in the diet was nearly completely absorbed and excreted in urine, with some fecal excretion. The hypothesized MOA for liver tumors induced by PHMB in drinking water is: 1) severe dehydration and starvation because of unpalatability, followed by ingestion with rapid absorption and urinary excretion; 2) increased hepatocyte proliferation; and 3) induction of hepatocellular foci and tumors. The PHMB-induced rat hepatocellular tumors are unlikely to pose a human cancer risk. However, the actual MOA has not been determined.

Low-dose dose-response for reduced cell viability after exposure of human keratinocyte (HEK001) cells to arsenite.

The in vitro arsenite (AsIII) cytotoxicity dose-response (DR) of human keratinocytes (HEK001) was examined at greater statistical resolution than ever previously reported using the MTT assay to determine cell viability. Fifty-four 96-well plates were treated with AsIII concentrations of 0.25, 0.5, 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, or 30 µM. Because of unexpected variation in viability response patterns, a two-stage DR analysis was used in which data on plate-specific viability (%), estimated as 100% times the ratio of measured viability in exposed to unexposed cells, were fit initially to a generalized lognormal response function positing that HEK001 cells studied consisted of: a proportion P of relatively highly sensitive (HS) cells, a proportion Po of relatively resistant cells, and a remaining (1-P-Po) fraction of typical-sensitivity (TS) cells exhibiting the intermediate level of AsIII sensitivity characteristic of most cells in each assay. The estimated fractions P and Po were used to adjust data from all 54 plates (and from the 28 plates yielding the best fits) to reflect the condition that P = Po = 0 to provide detailed DR analysis specifically for TS cells. Four DR models fit to the combined adjusted data were each very predictive (R2 > 0.97) overall but were inconsistent with at least one of the data set examined (p < 10-5). Adjusted mean responses at ≤3 µM were approximately equal (p > 0.30) and exceeded 100% significance (p ≤ 10-6). A low-dose hormetic model provided the best fit to the combined adjusted data for TS cells (R2 = 0.995). Marked variability in estimates of P (the proportion of apparent HS cells) was unexpected, not readily explained, and warrants further study using additional cell lines and assay methods, and in vivo.

A novel Aurora kinase A inhibitor MK-8745 predicts TPX2 as a therapeutic biomarker in non-Hodgkin lymphoma cell lines.

Selective small-molecule kinase inhibitors have encouraging clinical efficacy in several malignancies. These agents are still limited to a subset of patients, indicating the need to develop therapeutic biomarkers that influence clinical benefit. In this study, we demonstrate that treatment with MK-8745, a novel Aurora-A specific inhibitor, leads to cell cycle arrest at the G2/M phase with accumulation of tetraploid nuclei followed by cell death in non-Hodgkin lymphoma (NHL) cell lines. The sensitivity of the cell lines to MK-8745 is correlated with the expression level of Aurora-A activator. The siRNA knockdown of Aurora-A activator TPX2 (targeting protein for Xenopus kinase-like protein 2) increased MK-8745 sensitivity in less-MK-8745-sensitive NHL cell lines, whereas overexpression of TPX2 in high-MK-8745-sensitive NHL cell lines increased drug resistance. Our results indicate that TPX2 may serve as a biomarker for identifying subpopulations of patients sensitive to Aurora-A inhibitor treatment.

Identification of a novel TGFß/PKA signaling transduceome in mediating control of cell survival and metastasis in colon cancer.

BACKGROUND: Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFß tumor suppressor activities in the metastatic process is essentially unknown. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing in vitro and in vivo techniques, we have shown that loss of TGFß tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFß receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFß/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFß/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFß receptor restoration with reactivation of the TGFß/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival. CONCLUSION/SIGNIFICANCE: This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFß function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFß signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFß/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors.