The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress.

The ubiquitin-specific peptidase 10 (USP10) plays a context-specific, pro or anti-tumorigenic role in different malignancies. However, the role of USP10 in pancreatic cancer remains unclear. Our protein and RNA level analysis from archived specimens and public databases show that USP10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and expression correlates with poor overall patient survival. Phenotypically, silencing USP10 decreased viability, clonal growth and invasive properties of pancreatic cancer cells. Mechanistically, silencing USP10 upregulated BiP and induced endoplasmic reticulum (ER) stress that led to an unfolded protein response (UPR) and upregulation of PERK, IRE1α. Decreased cell viability of USP10 silenced cells could be rescued by a chemical chaperone that promotes protein folding. Our studies suggest that USP10 by protecting pancreatic cancer cells from ER stress may support tumor progression.

KRCC1, a modulator of the DNA damage response.

The lysine-rich coiled-coil 1 (KRCC1) protein is overexpressed in multiple malignancies, including ovarian cancer, and overexpression correlates with poor overall survival. Despite a potential role in cancer progression, the biology of KRCC1 remains elusive. Here, we characterize the biology of KRCC1 and define its role in the DNA damage response and in cell cycle progression. We demonstrate that KRCC1 associates with the checkpoint kinase 1 (CHK1) upon DNA damage and regulates the CHK1-mediated checkpoint. KRCC1 facilitates RAD51 recombinase foci formation and augments homologous recombination repair. Furthermore, KRCC1 is required for proper S-phase progression and subsequent mitotic entry. Our findings uncover a novel component of the DNA damage response and a potential link between cell cycle, associated damage response and DNA repair.

Physiologically Based Pharmacokinetic Modeling and Tissue Distribution Characteristics of SHetA2 in Tumor-Bearing Mice.

The orally available novel small molecule SHetA2 is the lead sulfur-containing heteroarotinoid that selectively inhibits cancer cells over normal cells, and is currently under clinical development for anticancer treatment and cancer prevention. The objective of this study was to assess and characterize the tissue distribution of SHetA2 in tumor-bearing mice by developing a physiologically based pharmacokinetic (PBPK) model. An orthotopic SKOV3 ovarian cancer xenograft mouse model was used to most accurately mimic the ovarian cancer tumor microenvironment in the peritoneal cavity. SHetA2 concentrations in plasma and 14 different tissues were measured at various time points after a single intravenous dose of 10 mg/kg and oral dose of 60 mg/kg, and these data were used to develop a whole-body PBPK model. SHetA2 exhibited a multi-exponential plasma concentration decline with an elimination half-life of 4.5 h. Rapid and extensive tissue distribution, which was best described by a perfusion rate-limited model, was observed with the tissue-to-plasma partition coefficients (kp = 1.4-21.2). The PBPK modeling estimated the systemic clearance (76.4 mL/h) from circulation as a main elimination pathway of SHetA2. It also indicated that the amount absorbed into intestine was the major determining factor for the oral bioavailability (22.3%), while the first-pass loss from liver and intestine contributed minimally (< 1%). Our results provide an insight into SHetA2 tissue distribution characteristics. The developed PBPK model can be used to predict the drug exposure at tumors or local sites of action for different dosing regimens and scaled up to humans to correlate with efficacy.

HER2/HER3 regulates lactate secretion and expression of lactate receptor mRNA through the MAP3K4 associated protein GIT1.

One of the major features of cancer is Otto Warburg's observation that many tumors have increased extracellular acidification compared to healthy tissues. Since Warburg's observation, the importance of extracellular acidification in cancer is now considered a hallmark of cancer. Human MAP3K4 functions upstream of the p38 and JNK mitogen activated protein kinases (MAPKs). Additionally, MAP3K4 is required for cell migration and extracellular acidification of breast cancer cells in response to HER2/HER3 signaling. Here, we demonstrate that GIT1 interacts with MAP3K4 by immunoprecipitation, while cellular lactate production and the capacity of MCF-7 cells for anchorage independent growth in soft agar were dependent on GIT1. Additionally, we show that activation of HER2/HER3 signaling leads to reduced expression of lactate receptor (GPR81) mRNA and that both, GIT1 and MAP3K4, are necessary for constitutive expression of GPR81 mRNA. Our study suggests that targeting downstream proteins in the HER2/HER3-induced extracellular lactate signaling pathway may be a way to inhibit the Warburg Effect to disrupt tumor growth.

Anti-CD73 and anti-OX40 immunotherapy coupled with a novel biocompatible enzyme prodrug system for the treatment of recurrent, metastatic ovarian cancer.

Approximately 75% of ovarian cancer is diagnosed once metastasis to the peritoneal cavity has occurred. A large proportion of patients eventually develop platinum-resistive tumors, which are considered terminal. In order to provide an alternative a novel fusion protein, mCTH-ANXA5, has been developed for the treatment of recurrent, metastatic ovarian cancer. The fusion protein combines annexin V (ANXA5), an ovarian tumor and tumor vasculature targeting protein, with mutated cystathionine gamma-lyase (mCTH), an enzyme that converts selenomethionine (SeMet) into toxic methylselenol, which generates reactive oxygen species and eventual tumor cell death. In order to further enhance the therapeutic efficacy, anti-CD73 and anti-OX40 immunostimulants were combined with mCTH-ANXA5, resulting in an increase of survival by 100% from 12 to 24 days post-therapy and decrease tumor burden in mice with orthotopic metastatic ovarian cancer. Further evaluation of the combination therapy revealed a strong antibody-mediated immune response, and an increased infiltration of cytotoxic T-cells along with a decrease in tumor promoting immune cells. This study demonstrates the efficacy of a synergistic, multi-drug system by attacking the tumor as well as enlisting the body's own defense system to treat the patient.

Bioanalytical method development and validation of HPLCUV assay for the quantification of SHetA2 in mouse and human plasma: Application to pharmacokinetics study.

BACKGROUND: SHetA2 is an oral anticancer agent being investigated for cancer treatment and prevention. The aim of this study was to develop and validate a simple, cost-effective, and sensitive HPLC-UV method for the quantification of SHetA2 in biological samples and to apply the method to pharmacokinetic studies of the drug. METHODS: Sample preparation for mouse and human plasmas involved liquid-liquid precipitation and extraction using chilled acetonitrile with 2, 3-Diphenylquinoxaline as an internal standard. The separation of SHetA2 and internal standard was achieved via Waters XBridge™ BEH 130 C18 (3.5 µm, 2.1×150 mm) column coupled with a Waters XBridge™ C-18 (3.5 µm, 2.1×10 mm) guard column using 65% v/v acetonitrile: distilled water as a mobile phase in an isocratic mode with a flow rate of 0.18 ml/min. The analytes were eluted at a detection wavelength of 341 nm at a column temperature of 25°C. RESULTS: The method was validated across a range of 5-1000 ng/ml for SHetA2 in plasma, with a lower limit of quantification of 5 ng/ml. The method showed high recovery in human (79.9-81.8%) and mouse (95.4-109.2%) plasma with no matrix effect. The intra- and inter-day accuracy and precision studies demonstrated that the method was specific, sensitive, and reliable. Stability studies showed that SHetA2 is stable for 20 h postoperatively in the auto sampler, and for six weeks at -80°C in plasma. Repetitive freezing and thawing may be avoided by preparing the aliquots and storing them at -80°C. The developed method was successfully applied to study the plasma pharmacokinetics of SHetA2 in tumor-bearing nude mice after intravenous and oral administration. CONCLUSION: A novel method for quantifying SHetA2 in mouse and human plasmas has been validated and is being applied for pharmacokinetic evaluation of SHetA2 in tumor-bearing mice. The developed method will be utilized for the quantification of SHetA2 in clinical studies.

Synthesis and evaluation of second generation Flex-Het scaffolds against the human ovarian cancer A2780 cell line.

Flexible Heteroarotinoids (Flex-Hets) are a class of substituted di-aryl compounds that exhibit potent anti-cancer activity without toxicity. They were derived from the more conformationally restricted, 2-atom linker Hets by substitution of the 2-atom linker with a 3-atom urea or thiourea linker, which conferred more potent inhibitory activity against cancer cell lines. The objectives of this structure activity relationship (SAR) study were to determine if a 4-atom acrylamide linker and various substitutions on the terminal aryl ring altered the anti-cancer activity of these second generation Flex-Het compounds compared to the parent Flex-Het compound, SHetA2, which has a thiourea linker and a nitro substituent. Biological activity was measured using a cytotoxicity assay of the human A2780 ovarian cancer cell line treated with a range of compound concentrations. Nitrogen-based substitutions on the terminal aryl group caused similar, but slightly reduced efficacies and potencies. Exceptions were systems that had a nitro group at the para position, the potencies of which were better than that of SHetA2 with efficacies that were only slightly reduced compared to SHetA2. Similarly, the potency of the system with a para dimethylamino group was greater than that of SHetA2. However, a 30% reduction in efficacy compared to SHetA2 was noted. While specific members with the 4-atom acrylamide linker did exhibit excellent potency, the efficacy was slightly below that of SHetA2. Thus, a gradient of activities was observed as the substituent on the aryl ring was altered.

Measurements of adiposity as clinical biomarkers for first-line bevacizumab-based chemotherapy in epithelial ovarian cancer.

OBJECTIVE: There is a lack of reliable indicators to predict who will benefit most from anti-angiogenic therapy, such as bevacizumab. Recognizing obesity is associated with increased levels of VEGF, the main target of bevacizumab, we sought to assess if adiposity, measured in terms of BMI, subcutaneous fat area (SFA), and visceral fat area (VFA) was prognostic. METHODS: Reviewed 46 patients with advanced EOC who received primary treatment with bevacizumab-based chemotherapy (N=21) or chemotherapy alone (N=25) for whom complete records, CT prior to the first cycle of chemo, and serum were available. CT was used to measure SFA and VFA by radiologists blinded to outcomes. ELISA was used to measure serum levels of VEGF and angiopoietin-2 in the bevacizumab group. RESULTS: BMI, SFA, and VFA were dichotomized using the median and categorized as "high" or "low". In the bevacizumab group median PFS was shorter for patients with high BMI (9.8 vs. 24.7months, p=0.03), while in the chemotherapy group median PFS was similar between high and low BMI (17.6 vs. 11.9months, p=0.19). In the bevacizumab group patients with a high BMI had higher median levels of VEGF and angiopoietin-2, 371.9 vs. 191.4pg/ml (p=0.05) and 45.9 vs. 16.6pg/ml (p=0.09) respectively. On multivariate analysis neither BMI, SFA, nor VFA were associated with PFS (p=0.13, p=0.86, p=0.16 respectively) or OS (p=0.14, p=0.93, p=0.28 respectively) in the chemotherapy group. However, in the bevacizumab group BMI was significantly associated with PFS (p=0.02); accounting for confounders adjusted HR for high vs. low BMI was 5.16 (95% CI 1.31-20.24). Additionally in the bevacizumab group SFA was significantly associated with OS (p=0.03); accounting for confounders adjusted HR for high vs. low SFA was 3.58 (95% CI 1.12-11.43). CONCLUSION: Results provide the first evidence in EOC that patients with high levels of adiposity may not derive benefit from bevacizumab and that measurements of adiposity are likely to be a useful biomarker.

Insulin exerts direct effects on carcinogenic transformation of human endometrial organotypic cultures.

Epidemiological studies suggest an association between elevated insulin levels and endometrial cancer. We studied the effects of insulin on normal endometrial cell proliferation with cytotoxicity assays. Organotypic cultures were used to determine the effects of insulin on the development of malignant histological features and anchorage independent growth. Western Blots were used to analyze the mitogen-activated protein kinases and AKT pathways. We found that insulin exerts direct effects on endometrial cells by increasing proliferation and promoting carcinogenesis. Our results suggest that this occurs through ERK 1/2 and glycogen synthase kinase-3ß Ser9 phosphorylation.

HER2/HER3 regulates extracellular acidification and cell migration through MTK1 (MEKK4).

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In this study we show MTK1 is required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced cell migration in MCF-7 breast cancer cells. We demonstrate that HRG stimulation leads to association of MTK1 with activated HER3 in MCF-7 and T-47D breast cancer cells. Activated HER3 association with MTK1 is dependent on HER2 activation and is decreased by pre-treatment with the HER2 inhibitor, lapatinib. Moreover, we also identify the actin interacting region (AIR) on MTK1. Disruption of actin cytoskeletal polymerization with cytochalasin D inhibited HRG induced MTK1/HER3 association. Additionally, HRG stimulation leads to extracellular acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.

CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS: Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 µM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS: Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS: These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.

Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK.

OBJECTIVES: Altered signalling in B cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signalling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterise the role of BANK1 and BLK in SLE, a genetic interaction analysis was performed hypothesising that genetic interactions could reveal functional pathways relevant to disease pathogenesis. METHODS: The GPAT16 method was used to analyse the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localisation, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK. RESULTS: Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, the possibility that BANK1 and BLK could also show a protein-protein interaction was tested. The co-immunoprecipitation and co-localisation of BLK and BANK1 were demonstrated. In a Daudi cell line and primary naive B cells endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies. CONCLUSION: This study shows a genetic interaction between BANK1 and BLK, and demonstrates that these molecules interact physically. The results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signalling pathway.

Expression of common chromosomal fragile site genes, WWOX/FRA16D and FHIT/FRA3B is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR.

Common chromosomal fragile sites are unstable genomic loci susceptible to breakage, rearrangement, and are highly recombinogenic. Frequent alterations at these loci in tumor cells led to the hypothesis that they may contribute to cancer development. The two most common chromosomal fragile sites FRA16D and FRA3B which harbor WWOX and FHIT genes, respectively, are frequently altered in human cancers. Here we report that environmental carcinogens, ultraviolet (UV) light, and Benzo[a]pyrene diol epoxide (BPDE), significantly downregulate expression of both genes. On the other hand, we observe that ionizing radiation (IR) does not affect expression of these genes, suggesting that the effect of repression exerted by UV and BPDE is not just a consequence of DNA damage but may be a result of different signaling pathways triggered by specific DNA lesions. Such downregulation correlates with an induction of an S-phase delay in the cell cycle. Treatment of UV-irradiated cells with caffeine abrogates the S-phase delay while concomitantly overcoming the repression phenomenon. This suggests the involvement of unique cell cycle checkpoint mechanisms in the observed repression. Therefore, it is hypothesized that protracted downregulation of the putative tumor suppressor genes WWOX and FHIT by environmental carcinogens may constitute an additional mechanism of relevance in the initiation of tumorigenesis.