Growth Of Established Tumors Research Articles

MicroRNA-185 regulates chemotherapeutic sensitivity in gastric cancer by targeting apoptosis repressor with caspase recruitment domain.

Gastric cancer remains the second leading cause of cancer deaths worldwide. Resistance to chemotherapy is a significant barrier for effective cancer treatment. Here, we identified miR-185 to be a contributor to chemosensitivity in gastric cancer. We observed low levels of miR-185 in gastric cancer cell lines and clinical tissues, compared with gastric epithelium cell line and noncancerous tissues. Furthermore, enforced expression of miR-185 increased the sensitivity of gastric cancer cells to low-dose chemotherapeutic agents, which alone cannot trigger significant apoptosis. Conversely, knockdown of endogenous miR-185 prevented high-dose chemotherapy-induced apoptosis. In elucidating the molecular mechanism by which miR-185 participated in the regulation of chemosensitivity in gastric cancer, we discovered that apoptosis repressor with caspase recruitment domain (ARC) is a direct target of miR-185. The role of miR-185 was confirmed in gastric tumor xenograft model. The growth of established tumors was suppressed by a combination therapy using enforced miR-185 expression and a low dose of anticancer drugs. Finally, we found that RUNX3 (Runt-related transcription factor) was involved in the activation of miR-185 at the transcriptional level. Taken together, our results reveal that RUNX3, miR-185 and ARC regulate the sensitivity of gastric cancer cells to chemotherapy.

VISTA Regulates the Development of Protective Antitumor Immunity

V-domain Ig suppressor of T-cell activation (VISTA) is a novel negative checkpoint ligand that is homologous to PD-L1 and suppresses T-cell activation. This study demonstrates the multiple mechanisms whereby VISTA relieves negative regulation by hematopoietic cells and enhances protective antitumor immunity. VISTA is highly expressed on myeloid cells and Foxp3(+)CD4(+) regulatory cells, but not on tumor cells within the tumor microenvironment (TME). VISTA monoclonal antibody (mAb) treatment increased the number of tumor-specific T cells in the periphery and enhanced the infiltration, proliferation, and effector function of tumor-reactive T cells within the TME. VISTA blockade altered the suppressive feature of the TME by decreasing the presence of monocytic myeloid-derived suppressor cells and increasing the presence of activated dendritic cells within the tumor microenvironment. In addition, VISTA blockade impaired the suppressive function and reduced the emergence of tumor-specific Foxp3(+)CD4(+) regulatory T cells. Consequently, VISTA mAb administration as a monotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studies explored a combinatorial regimen using VISTA blockade and a peptide-based cancer vaccine with TLR agonists as adjuvants. VISTA blockade synergized with the vaccine to effectively impair the growth of established tumors. Our study therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or in combination with additional immune-targeted strategies for cancer immunotherapy.

Gemcitabine chemotherapy induces phenotypic alterations of tumor cells that facilitate antitumor T cell responses in a mouse model of oral cancer

Gemcitabine (GEM) is a pyrimidine nucleoside analogue that is a new chemotherapeutic agent used for treating various cancers. Because accumulating evidence indicates that GEM may activate host immune responses, its potential as an immune modulator in cancer chemotherapy has generated considerable interest. In the present study, we investigated the antitumor effects of GEM using a mouse oral cancer model using immunological analyses. We examined apoptotic cell death of tumor cells with GEM treatment both in vitro and in vivo. We also investigated whether in vivo administration of GEM affected the distributions of immune cells, tumor-cell surface expression levels of immune accessory molecules and T cell immune responses in tumor-bearing mice. GEM induced significant oral cancer-cell apoptosis in vitro, and in vivo GEM administration markedly attenuated established mouse tumor growth. In vivo GEM administration decreased the numbers of both myeloid-derived suppressor cells (MDSCs) and B cells in tumor-bearing mice and enhanced dendritic cell maturation. Moreover, GEM treatment upregulated tumor-cell surface expressions of several immune accessory molecules and adhesion molecules, including CD80, CD86, CD40, ICAM-1, VCAM-1, and P-selectin. Remarkably, these tumor cells augmented tumor specific T-cell responses. These results suggest that GEM can induce host antitumor immune responses, which would facilitate antitumor effects in the treatment of oral cancer.

Intracellular Delivery of ERBB2 siRNA and p53 Gene Synergistically Inhibits the Growth of Established Tumor in an Immunocompetent Mouse

Breast cancer is one of the leading causes of deaths worldwide in women with hormone therapy, chemotherapy, targeted therapies, or their combinations being the current options for treating the disease at the different stages (stages I-III) with associated side-effects or increasing life-span at the advanced stage (stage IV). Small interfering RNA (siRNA) as an effective tool to selectively knockdown of a particular gene could be harnessed in combination with plasmid DNA (carrying a gene of interest) and conventional anti-cancer drugs for precisely treating breast cancer with minimal side effects. However the limitation of the naked siRNA and DNA in penetrating the plasma membrane and their sensitiveness to nuclease-mediated cleavage render the technology rather complex in therapeutic intervention. Recently, we have developed pH-sensitive carbonate apatite as a potential nano-carrier to efficiently deliver siRNA or DNA across the cell membrane and facilitate them to escape endosomal acidic compartment resulting in specific cleavage of a particular mRNA transcript or expression of a desirable protein, respectively. Moreover, we demonstrated nanoparticle-assisted delivery of the siRNAs targeting cyclin B1, PLCgamma- 2/calmodulin1, NFκB1/NFκB2, ABCG2/ABCB1 and cROS1 mRNAs sensitizes cervical adenocarcinoma and breast cancer cells towards traditional anti-cancer drugs. Here, we report that co-delivery of the siRNA targeting HER2/ErbB2 gene transcript and p53 gene with the help of carbonate apatite nanoparticles synergistically induces inhibition of growth/proliferation of breast cancer cell lines as well as regression of the breast tumor induced in Balb/c mice. Additionally, combined delivery of nanoparticle-associated HER2/ErbB2 siRNA and p53 gene apparently slows down the growth of the established tumor in presence of doxorubicin or paclitaxel compared with the individual free drugs. Thus, the combination of HER2/ErbB2 knockdown and restoring of normal p53 function could be a highly promising approach necessitating further studies through pre-clinical trials with different models of breast cancer to establish the therapeutic role of this combination therapy prior to conducting clinical trials in breast cancer patients.

Doubling Down on the PI3K-AKT-mTOR Pathway Enhances the Antitumor Efficacy of PARP Inhibitor in Triple Negative Breast Cancer Model beyond BRCA-ness

Phosphoinositide 3-kinase (PI3K) pathway, in addition to its pro-proliferative and antiapoptotic effects on tumor cells, contributes to DNA damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mammalian target of rapamycin (mTOR) inhibitor, would induce an efficient antitumor effect in BRCA-competent triple negative breast cancer (TNBC) model when combined with ABT888 and carboplatin. Mechanism-based in vitro studies demonstrated that GDC-0980 treatment alone or in combination led to DNA damage (increased pγH2AXS139; Western blot, immunofluorescence), gain in poly ADP-ribose (PAR), and a subsequent sensitization of BRCA-competent TNBC cells to ABT888 plus carboplatin with a time-dependent 1) decrease in proliferation signals (pAKTT308/S473, pP70S6KT421/S424, pS6RPS235/236), PAR/poly(ADP-ribose) polymerase (PARP) ratios, PAR/pγH2AX ratios, live/dead cell ratios, cell cycle progression, and three-dimensional clonogenic growths and 2) increase in apoptosis markers (cleaved caspases 3 and 9, a pro-apoptotic BH3-only of Bcl-2 family (BIM), cleaved PARP, annexin V). The combination was effective in vitro in BRCA-wild-type PIK3CA-H1047R-mutated BT20 and PTEN-null HCC70 cells. The combination blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, CD31, phosphorylated vascular endothelial growth factor receptor, pS6RPS235/236, and p4EBP1T37/46 as well as an increase in cleaved caspase 3 immunohistochemistry (IHC) levels. Interestingly, a combination with GDC-0941, a pan-PI3K inhibitor, failed to block the tumor growth in MDA-MB231. Results demonstrate that the dual inhibition of PI3K and mTOR regulates DDR. In a BRCA-competent model, GDC-0980 enhanced the antitumor activity of ABT888 plus carboplatin by inhibiting both tumor cell proliferation and tumor-induced angiogenesis along with an increase in the tumor cell apoptosis. This is the first mechanism-based study to demonstrate the integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor action of PARP inhibitor in TNBC. [Display omitted] [Display omitted] [Display omitted] [Display omitted]

Abstract P3-04-02: Absence of PTEN facilitates the anti-tumor efficacy of GDC-0980 in combination with ABT888 plus carboplatin in BRCA1-competent triple negative breast cancer

Abstract Introduction: PI3K pathway, in addition to its pro-proliferative and anti-apoptotic effects on tumor cells, is known to contribute to DNA-damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mTOR inhibitor, will induce an efficient anti-tumor effect in BRCA-competent PTEN-null TNBC cells when combined with PARP inhibitor, ABT888 and carboplatin. We propose that in PTEN-null BRCA-competent TNBC model, the growth of TNBC tumor will be blocked due to the inhibition of (1) HR and NHEJ and (2) PI3K-mTOR pathway mediated survival signals following treatment with GDC-0980, when combined with PARP inhibitor (impaired DNA-SSB-repair) and carboplatin (increased DNA-DSB). Purpose: Here we tested the efficacy of a combination of GDC-0980 with ABT888 plus carboplatin in BRCA-competent PTEN-null model of TNBC. Methods: Athymic mice bearing PTEN-null TNBC xenograft tumors were treated with GDC-0980 alone or in combination with ABT888 and carboplatin. Results: Dual inhibition of PI3K and mTOR by GDC-0980 alone as well as in the presence of carboplatin plus ABT888 changed the state of the repair of DNA-damage in BRCA-competent PTEN null TNBC cells, which led to increased cellular apoptotic signals in addition to decreased survival/proliferative signals. GDC-0980 treatment led to DNA damage (increased pgH2AX), gain in PAR and a subsequent sensitization of BRCA-competent PTEN-null MDA-MB468 TNBC cells to ABT888 plus carboplatin with a time-dependent (1) decrease in proliferation signals (pAKT T308/S473, pP70S6K, pS6RP), PAR/PARP ratios, PAR/pgH2AX ratios, live/dead cell ratios, cell-cycle progression and clonogenic 3D growth and (2) increase in apoptosis markers (cleaved-caspase 3, 9, BIM, cleaved-PARP and annexinV positivity). These effects are more pronounced in MDA-MB468 than in RAS/RAF mutated MDA-MB231 cells. GDC-0980 alone and in combination with ABT888 plus carboplatin inhibited cell cycle progression, increased apoptosis, and decreased live/dead cell ratios in BRCA-competent PTEN null TNBC cells. GDC-0980 alone and in combination with ABT888 plus carboplatin attenuated anchorage -dependent and -independent clonogenic 3D growth comparatively more in BRCA-competent PTEN-null cells TNBC cells than MDA-MB231 cells. GDC-0980 in combination with ABT888 plus carboplatin blocked the growth of established PTEN-null TNBC tumors as compared to vehicle control(s) with a concomitant decrease in tumor Ki67 and CD31 IHC-stains. Conclusion: This is the first mechanism-based study to demonstrate that in BRCA-competent PTEN-null TNBC model, GDC-0980 enhanced antitumor activity of ABT888, in the presence of carboplatin by inhibiting DDR system in conjunction with the inhibition of PI3K-mTOR pathway-mediated proliferative, and anti-apoptotic signals. Considering (1) the importance of PARP as the target in TNBC, (2) the existence of a large percentage of BRCA-competent TN and/or basal type BC patients and (3) the high frequency of PTEN-null-ness in this subset of BC, this combination merits further investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-02.

Abstract P5-06-01: The PI3K inhibitor GDC-0941 combines with trastuzumab for superior anti-tumor efficacy in HER2+ breast cancer models

Abstract Background: PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Purpose: Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Experimental Design: The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Clonogenic growth was tested by 3D ON-TOP assay and apoptosis markers were also tested. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results: (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC50's ranging from 0.35 μM to 1 μm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (3) GDC-0941 treatment increased apoptosis markers (CL-CASPASE3 and annexinV positivity), (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) in vivo, the combination of GDC-0941 and T significantly reduced established tumor growth in both sensitive (82%) and resistant (79%) models with concomitant decrease of different PD markers. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical efficacy of T therapy, as well as to prevent or delay the development of resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-06-01.

Identifying the optimal dose of ritonavir in the treatment of malignancies

Identifying the optimal dose of ritonavir therapy overcomes the chemical resistance may exhibit in some cases due to poor prognosis of imprecise staging. Dose modeling was performed by analyzing previously published data of ritonavir cancer growth inhibition in vitro and in vivo. In-vitro 3H-Thymidine-based cell proliferation assay was performed on samples of the GL15 cell line incubated with 0, 1, 10 and 100 μ M of ritonavir. Proliferation inhibition was quantified to identify energy of the used doses as described before in earlier studies. Models involving in-vivo growth of established breast cancer tumor (MDA-MB-231), KSIMM tumor and EL4-T cell thymomas in mice were used. The effects of 40 mg/kg/day for 52 days, 30 mg/kg/day for 15 days and 8.8 mg/mouse/day for about 1 week of ritonavir in those xenograft growths respectively were monitored and quantified to identify energy of those doses as described before in earlier studies. Ritonavir demonstrated an in-vitro reduction in proliferation rate in dose dependent manner. The energy of the in-vitro influences following ritonavir therapy were perfectly correlated (r = 1) with ritonavir dose, allowed to establish an efficient energy-model with a perfect fit (R2=1) describes the energy yield by ritonavir doses, enables to administer the appropriate dose. Ritonavir had also a significant influence in-vivo on all sizes of treated tumors compared to the control animals such that the energy yield by the administered drug as derived from the energy-model was 100% identical to the induced influence in tumor energy. The in-vitro determination of inhibition to proliferation by ritonavir doses is useful to characterize the response of cancer to ritonavir therapy targeting patient-personalized cancer medicine. The molecular method of response determination by 3H-TDR incorporation and ritonavir dose-energy model are reliable to avoid chemo-resistance by identifying the optimal dosing regimens and schedules prior therapy allowing the use of much lower dose of ritonavir and thus decreases the drug side effects and risks of relapse.

Enhanced antitumor immunotherapeutic effect of B-cell-based vaccine transduced with modified adenoviral vector containing type 35 fiber structures

For successful clinical tumor immunotherapy outcomes, strong immune responses against tumor antigens must be generated. Cell-based vaccines compromise one strategy with which to induce appropriate strong immune responses. Previously, we established a natural killer T-cell (NKT) ligand-loaded, adenoviral vector-transduced B-cell-based anticancer cellular vaccine. To enhance tumor antigen delivery to B cells, we established a modified adenoviral vector (Ad-k35) that encoded a truncated form of the breast cancer antigen Her2/neu (Ad-k35HM) in which fiber structure was substituted with adenovirus serotype 35. We observed increased tumor antigen expression with Ad-k35HM in both human and murine B cells. In addition, an Ad-k35HM-transduced B-cell vaccine elicited strong antigen-specific cellular and humoral immune responses that were further enhanced with the additional loading of soluble NKT ligand KBC009. An Ad-k35HM-transduced, KBC009-loaded B-cell vaccine efficiently suppressed the in vivo growth of established tumors in a mouse model. Moreover, the vaccine elicited human leukocyte antigen (HLA)-A2 epitope-specific cytotoxic T-cell responses in B6.Cg (CB)-Tg (HLA-A/H2-D) 2Enge/Jat mice. These findings indicated that the Ad-k35 could be appropriate for the preclinical and clinical development of B-cell-based anticancer immunotherapies.

A lovastatin-elicited genetic program inhibits M2 macrophage polarization and enhances T cell infiltration into spontaneous mouse mammary tumors

Beyond their ability to inhibit cholesterol biosynthesis, the statins have pleiotropic effects that include anti-inflammatory and immunomodulatory activities. Statins could have clinical utility, alone or in combination with other chemotherapeutics, in the treatment of cancer. The mechanisms that underlie the anti-tumor activity of the statins are nonetheless poorly defined. No studies have analyzed how they alter the tumor-associated leukocyte infiltrate, a central factor that influences tumor stroma and cancer evolution. Here we used HER2/neu transgenic (Tg-neu) mice to analyze the effect of lovastatin (Lov) on the inflammatory reaction of spontaneous mammary tumors. Lov treatment of tumor-bearing Tg-neu mice did not alter growth of established tumors, but significantly reduced the number of new oncogenic lesions in these mice. Moreover, Lov inhibited the growth of newly implanted Tg-neu tumors in immunocompetent but not in immunodeficient mice. We found that Lov enhanced tumor infiltration by effector T cells, and reduced the number of immunosuppressive and pro-angiogenic M2-like tumor-associated macrophages (TAM). Concomitantly, the drug improved the structure and function of the tumor vasculature, measured as enhanced tumor oxygenation and penetration of cytotoxic drugs. Microarray analysis identified a Lov-elicited genetic program in Tg-neu tumors that might explain these effects; we observed Lov-induced downregulation of placental growth factor, which triggers aberrant angiogenesis and M2-like TAM polarization. Our results identify a role for lovastatin in the shaping and re-education of the inflammatory infiltrate in tumors, with functional consequences in angiogenesis and antitumor immunity.

Abstract C04: HGFL promotes the development and progression mammary tumors and represents an attractive therapeutic target

Abstract A standard therapy for breast cancer is the use of receptor tyrosine kinase (RTK) inhibitors. RTKs efficiently target multiple receptors, including the Ron transmembrane receptor, whose activation leads to increased survival, migration, and angiogenesis through signaling pathways such as PI3K/Akt and NF-κB. Ron overexpression is correlated with increased metastasis and poor outcomes in patients. The only ligand for Ron is hepatocyte growth factor-like protein (HGFL), which is an endocrine factor secreted into the circulation from the liver. Published reports demonstrated that ectopic overexpression of HGFL in cancer cells leads to increased metastasis. However, no studies have examined the role of endogenous HGFL in Ron activation during tumor development and metastasis. We sought to test the hypothesis that HGFL is required for Ron receptor activation and mammary tumorigenesis. We utilized mice with mammary-specific Ron overexpression (MMTV-Ron), which develop mammary tumors with 100% incidence. MMTV-Ron mice were crossed to HGFL deficient (HGFL-/-) mice to generate MMTV-Ron X HGFL-/- mice. We compared mammary hyperplasia and tumor latency in MMTV-Ron mice with and without HGFL serially at 2.5, 4, 6, 8 and 10 months. Additional mice were followed to established tumor kinetics and growth and to examine metastatic dissemination. MMTV-Ron X HGFL-/- mice displayed decreased mammary hyperplasia and a significant delay in the time to tumor formation compared to control mice. Kinase assays show that Ron activation is reduced in mice with HGFL loss. Tumor cell proliferation was different between the groups while markers of apoptosis (TUNEL IHC and cleaved caspase 3 expression) were significantly increased in MMTV-Ron X HGFL-/- mice compared to controls. Western blot analysis of tumor lysates showed diminished activation of NF-κB signaling associated with HGFL loss. In conclusion, our data show that HGFL promotes the development of mammary hyperplasia and mammary tumor formation by enhancing tumor cell survival, Ron activation and NF-κB signaling. These data suggest that HGFL represents an excellent target for inhibiting Ron mediated tumor growth in human disease. Citation Format: Nancy M. Benight, Purnima Wagh, Susan E. Waltz. HGFL promotes the development and progression mammary tumors and represents an attractive therapeutic target. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C04.

P09 Therapeutic potential of NOSH-aspirin, a dual nitric oxide- and hydrogen sulfide-donating hybrid in colon cancer

Colorectal cancer is one of the major causes of cancer deaths worldwide. In patients with advanced disease, the efficacy of currently available treatments is limited. Nonsteroidal anti-inflammatory drugs (NSAIDs) in general and aspirin (ASA) in particular are prototypical chemopreventive agents. However, the use of NSAIDs is limited by their significant toxicity (gastrointestinal, renal, and cardiovascular). In our search for a “better NSAID”, we have developed NOSH-aspirin for the treatment of colon cancer. The notion that a modified NSAID maybe used to treat cancer is radical. NOSH-aspirin (NBS-1120) is a hybrid molecule that releases nitric oxide (NO) and hydrogen sulfide (H 2 S), two gasotransmitters of physiological relevance. NOSH-aspirin is the first NSAID-based agent that is active against colon cancer in the nano-molar range, is devoid of gastrointestinal and cellular toxicity, preferentially affects cancer cells compared to normal cells, and has a pleotropic mechanism of action. In vitro, NOSH-aspirin inhibited the growth of HT-29, HCT 15, and SW480 human colon cancer cell lines with IC 50 values of 48 ± 3, 50 ± 5, and 60 ± 4 nM at 24 h, respectively. The corresponding IC 50 for aspirin in all 3 cell lines was > 5,000,000 nM. This represents an enhanced potency of > 80,000-fold in the 3 cell lines at 24 h. Using HT-29 colon cancer cells, NOSH-aspirin had IC 50 s for cell growth inhibition of 20 nM at 48 h and 8 nM at 72 h. The corresponding values for aspirin were, 2,500,000 nM and 2,000,000 nM, respectively. This represents potency enhancements of ∼125–000-fold and ∼250,000-fold. Although highly potent, NOSH-aspirin was without any cytotoxicity as determined by lactate dehydrogenase release. Inhibiting NO and H 2 S release from NOSH-aspirin, increased the IC 50 for cell growth inhibition to ∼800,000 nM at 24 h. NOSH-aspirin had a synergistic effect with 5-flurouracil (5-FU), in HT-29 cells its IC 50 for cell growth inhibition at 24 h went down from 50 nM to 4 nM in presence of sub-toxic levels of 5-FU. Mechanistically, NOSH-aspirin dose-dependently induced apoptosis; inhibited proliferation; caused a G 0 /G 1 cell cycle block; increased reactive oxygen species (ROS); inhibited NF-#kappa B, regulating it by reciprocal S-nitrosylation and S-sulfhydration; and inhibited cytokine-induced NOS2 expression. In vivo, using a xenograft model of colon cancer, NOSH-aspirin dose-dependently reduced the growth of established tumors, reduced proliferation, induced apoptosis, reduced NF-#kappa B, and induced ROS. NOSH-aspirin also exhibited potent anti-inflammatory properties, inhibiting both cyclo-oxygense (COX)-1 and COX-2, thus reducing prostaglandin (PG) E 2 levels in a carrageenan-induced rat paw edema model. In conclusion, NOSH-aspirin is a promising candidate against cancer modulating key parameters that regulate cellular mass.

A Genome-wide siRNA Screen Identifies Proteasome Addiction as a Vulnerability of Basal-like Triple-Negative Breast Cancer Cells

Basal-like triple-negative breast cancers (TNBCs) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes: basal-like BPLER and myoepithelial HMLER. Expression of the screen's 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal, and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence.

Novel Recombinant Human B7-H4 Antibodies Overcome Tumoral Immune Escape to Potentiate T-Cell Antitumor Responses

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging. B7-H4 expression was detected on the surface of all fresh primary human tumors and tumor xenotransplants, but not on most established cell lines, and B7-H4 was lost rapidly by tumor xenograft cells after short-term in vitro culture. These results indicated an in vivo requirement for B7-H4 induction and defined conditions for targeting studies. To generate anti-B7-H4-targeting reagents, we isolated antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library derived from patients with ovarian cancer. We identified anti-B7-H4 scFv that reversed in vitro inhibition of CD3-stimulated T cells by B7-H4 protein. Notably, these reagents rescued tumor antigen-specific T-cell activation, which was otherwise inhibited by coculture with antigen-loaded B7-H4+ APCs, B7-H4+ tumor cells, or B7-H4- tumor cells mixed with B7-H4+ TAMs; peritoneal administration of anti-B7-H4 scFv delayed the growth of established tumors. Together, our findings showed that cell surface expression of B7-H4 occurs only in tumors in vivo and that antibody binding of B7-H4 could restore antitumor T-cell responses. We suggest that blocking of B7-H4/B7-H4 ligand interactions may represent a feasible therapeutic strategy for ovarian cancer.

Sorafenib/Regorafenib and Phosphatidyl Inositol 3 Kinase/Thymoma Viral Proto-Oncogene Inhibition Interact to Kill Tumor Cells

The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3-green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by ∼40%. In vivo, sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrate that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo.

Genetically modified T cells targeting neovasculature efficiently destroy tumor blood vessels, shrink established solid tumors and increase nanoparticle delivery

Converting T cells into tumor cell killers by grafting them with a chimeric antigen receptor (CAR) has shown promise as a cancer immunotherapeutic. However, the inability of these cells to actively migrate and extravasate into tumor parenchyma has limited their effectiveness in vivo. Here we report the construction of a CAR containing an echistatin as its targeting moiety (eCAR). As echistatin has high binding affinity to αvβ3 integrin that is highly expressed on the surface of endothelial cells of tumor neovasculature, T cells engrafted with eCAR (T-eCAR) can efficiently lyse human umbilical vein endothelial cells and tumor cells that express αvβ3 integrin when tested in vitro. Systemic administration of T-eCAR led to extensive bleeding in tumor tissues with no evidence of damage to blood vessels in normal tissues. Destruction of tumor blood vessels by T-eCAR significantly inhibited the growth of established bulky tumors. Moreover, when T-eCAR was codelivered with nanoparticles in a strategically designed temporal order, it dramatically increased nanoparticle deposition in tumor tissues, pointing to the possibility that it may be used together with nanocarriers to increase their capability to selectively deliver antineoplastic drugs to tumor tissues.

Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases

BackgroundMelanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics.MethodsWe used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development.ResultsWe confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition.ConclusionPEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment.

The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.

Local Administration of TLR Ligands Rescues the Function of Tumor-Infiltrating CD8 T Cells and Enhances the Antitumor Effect of Lentivector Immunization

Cancer vaccines, to date, have shown limited effect to control the growth of established tumors due largely to effector failure of the antitumor immune responses. Tumor lesion is characterized as chronic indolent inflammation in which the effector function of tumor-infiltrating lymphocytes (TILs) is severely impaired. In this study, we investigated whether the effector function of CD8 TILs could be rescued by converting the chronic inflammation milieu to acute inflammation within tumors. We found that injection of TLR3/9 ligands (polyI:C/CpG) into a tumor during the effector phase of lentivector (lv) immunization effectively rescued the function of lv-activated CD8 TILs and decreased the percentage of T regulatory within the tumor, resulting in a marked improvement in the antitumor efficacy of lv immunization. Mechanistically, rescue of the effector function of CD8 TILs by TLR3/9 ligands is most likely dependent on production, within a tumor, of type-1 IFN that can mature and activate tumor-infiltrating dendritic cells. The effector function of CD8 TILs could not be rescued in mice lacking intact type I IFN signaling. These findings have important implications for tumor immunotherapy, suggesting that type I IFN-mediated activation of tumor-infiltrating dendritic cells within a tumor will most likely restore/enhance the effector function of CD8 TILs and thus improve the antitumor efficacy of current cancer vaccines.

A Novel Benzothiazole Derivative YLT322 Induces Apoptosis via the Mitochondrial Apoptosis Pathway In Vitro with Anti-Tumor Activity in Solid Malignancies

Benzothiazole derivatives are known for various biological activities, and their potency in cancer therapy has received considerable attention in recent years. YLT322, a novel synthesized benzothiazole derivative, exhibits potent anti-tumor activity via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT322 showed growth inhibition against a broad spectrum of human cancer cells and induced apoptosis of HepG2 cells in a dose- and time-dependent manner. The occurrence of its apoptosis was associated with activation of caspases-3 and -9, but not caspase-8. YLT322 increased the expression of Bax, decreased the expression of Bcl-2, and induced the release of cytochrome c which activates the mitochondrial apoptotic pathway. The down-regulation of phosphorylated p42/44 MAPK and phosphorylated Akt was also observed. Moreover, YLT322 suppressed the growth of established tumors in xenograft models in mice without obvious side effects. Histological and immunohistochemical analyses revealed an increase in TUNEL and caspase-3-positive cells and a decrease in Ki67-positive cells upon YLT322. These results suggest that YLT322 may be a potential candidate for cancer therapy.