Targeted DeSUMOylation as a therapeutic strategy for multiple sclerosis.

SUMO (small ubiquitin like modifier) conjugated proteins have emerged as an important post translational modifier of cellular function. SUMOylation modulates several cellular processes involved in transcriptional regulation of genes, protein-protein interactions and DNA damage and repair. Since abnormalities in SUMOylation has been observed in neoplastic and neurodegenerative disorders, the SUMO pathway has become an attractive site for targeting of new therapies to regulate SUMOylation and reduce disease burden. Conjugation of SUMO to their respective substrates is orchestrated by an enzymatic cascade involving three main enzymes, E1, activation enzyme, E2, conjugating enzyme and E3, a protein ligase. Each of these enzymes are therefore potential "druggable" sites for future therapeutics. SUMOylation is a well-known mechanism by which the innate immune response is regulated in response to viral infections and in the adaptive immune response to tumor immunity. We have shown that small molecules which inhibit the SUMO activation pathway are also capable of inhibiting autoimmune response. TAK981 which forms adducts with SUMO and anacardic acid which inhibits the E1 enzyme of the SUMO pathway were effective in preventing the development of experimental allergic encephalitis (EAE), a mouse model of multiple sclerosis. Anacardic acid and TAK981 inhibited activation of TH17 cells and reduced clinical and pathological injury in IL-17 mediated myelin oligodendrocyte glycoprotein (MOG) induced EAE. Ginkgolic acid, another known inhibitor of SUMO pathway, was also shown to be effective in reducing the severity of inflammatory arthropathies which is also IL-17 mediated. In addition, the increase in the transcription of myelin genes with TAK981 and anacardic acid improved remyelination in experimental models of demyelination. In the present review paper, we examine the mechanism of action of inhibitors of the SUMO pathway on regulating the immune response and the possibility of the use of these agents as therapeutics for MS.

PD-1H/VISTA mediates immune evasion in acute myeloid leukemia.

Acute myeloid leukemia (AML) presents a pressing medical need in that it is largely resistant to standard chemotherapy as well as modern therapeutics, such as targeted therapy and immunotherapy, including anti-programmed cell death protein (anti-PD) therapy. We demonstrate that programmed death-1 homolog (PD-1H), an immune coinhibitory molecule, is highly expressed in blasts from the bone marrow of AML patients, while normal myeloid cell subsets and T cells express PD-1H. In studies employing syngeneic and humanized AML mouse models, overexpression of PD-1H promoted the growth of AML cells, mainly by evading T cell-mediated immune responses. Importantly, ablation of AML cell-surface PD-1H by antibody blockade or genetic knockout significantly inhibited AML progression by promoting T cell activity. In addition, the genetic deletion of PD-1H from host normal myeloid cells inhibited AML progression, and the combination of PD-1H blockade with anti-PD therapy conferred a synergistic antileukemia effect. Our findings provide the basis for PD-1H as a potential therapeutic target for treating human AML.

LAIR-1 agonism as a therapy for acute myeloid leukemia.

Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.

Inhibition of SUMOylation promotes remyelination and reduces IL-17 mediated autoimmune inflammation: Novel approach toward treatment of inflammatory CNS demyelinating disease.

Small ubiquitin like modifiers (SUMO) are reversible posttranslational modifiers of intracellular proteins. In the CNS, expression of myelin genes is regulated by state of SUMOylation of their respective transcription factors. In the immune system, deSUMOylation activates innate immune responses and promotes anti-viral immunity. However, the role played by SUMO in an adaptive immune response and in the development of T cell mediated autoimmune disease has not been previously described. TAK981 is a synthetic small molecule which by forming adducts with SUMO proteins prevents SUMOylation. We examined the expression of myelin genes and their transcription factors following culture with TAK981 in Oligodendrocyte Precursor Cells (OPC). We found that myelin basic protein (MBP), a key myelin protein, is upregulated in OPC in the presence of TAK981. We also found increased expression of transcription factors Sox10 and Myrf, which engage in the expression of MBP. In the Cuprizone model of demyelination/remyelination, animals which were treated with TAK981 showed increased remyelination in areas of demyelination and an increase in the number of maturing oligodendrocytes compared to vehicle treated controls. In in vitro cultures of lymphocytes, TAK981 reduced the expression of TH17 in T cells in mice immunized with MOGp35-55. Following in vivo treatment with TAK981, there was a significant reduction in the clinical and pathological severity in mice immunized to develop experimental allergic encephalitis (EAE). The dual effects of deSUMOylation on remyelination and in regulating an autoimmune adaptive response offers a novel approach to the management of human inflammatory demyelinating diseases such as multiple sclerosis.

Overexpression of microRNA-145 inhibits tumorigenesis through autophagy in chemotherapy and radiation resistant neuroblastoma cells.

MicroRNA-145 (miR-145) plays a suppressive role in the process of tumorigenesis and an important role in induction of autophagy. However, the exact role of miR-145 in therapeutically resistant neuroblastoma cells remain elusive. Herein, we sought to evaluate the effects of miR-145 overexpression in chemo­ and radiation-resistant neuroblastoma cells. We hypothesized that miR-145 affects the aggressiveness of resistant cells by enhancing autophagy. We established Cisplatin-resistant (CDDP-R), Vincristine-resistant (Vin-R), and radiation-resistant (Rad-R) neuroblastoma cells and found that miR-145 expression was significantly decreased in the resistant cells compared to the parental cells. Exogenously expression of miR-145 inhibited oncogenic properties such as proliferation, clonogenicity, anchorage-independent growth, cell migration, and tubule formation in the resistant cells. In addition, we also found that an autophagy protein marker, LC3, was only minimally expressed in the resistant cells. In particular, when miR-145 was overexpressed in the resistant cells, LC3 I and II were expressed and an increased punctate fluorescence of LC3 protein was found indicating the induction of autophagy. Taken together, our data suggests that miR-145 inhibits tumorigenesis and aggressiveness via modulation of autophagy in neuroblastoma.

Dual-Targeting AKT2 and ERK in cancer stem-like cells in neuroblastoma.

Neuroblastoma remains one of the most difficult pediatric solid tumors to treat. In particular, the refractory and relapsing neuroblastomas are highly heterogeneous with diverse molecular profiles. We previously demonstrated that AKT2 plays critical roles in the regulation of neuroblastoma tumorigenesis. Here we hypothesize that targeting AKT2 could block the signal transduction pathways enhanced in chemo- and/or radiation-resistant neuroblastoma cancer stem-like cells. We found cell proliferation and survival signaling pathways AKT2/mTOR and MAPK were enhanced in cisplatin (CDDP)- and radiation-resistant neuroblastoma cells. Blocking these two pathways with specific inhibitors, CCT128930 (AKT2 inhibitor) and PD98059 (MEK inhibitor) decreased cell proliferation, angiogenesis, and cell migration in these resistant cells. We further demonstrated that the resistant cells had a higher sphere-forming capacity with increased expression of stem cell markers CD133, SOX2, ALDH, Nestin, Oct4, and Nanog. Importantly, the tumorsphere formation, which is a surrogate assay for self-renewal, was sensitive to the inhibitors of AKT2 and MAPK. Taken together, our findings suggest that CDDP- and radiation-resistant cancer stem-like neuroblastoma cells might serve as a useful tool to improve the understanding of molecular mechanisms of therapeutic resistance. This may aid in the development of more effective novel treatment strategies and better clinical outcomes in patients with neuroblastoma.

Identification of α-N-catenin as a novel tumor suppressor in neuroblastoma.

The lost expression of α-catenin has been found in cancers, and reinstalling α-catenin inhibits tumor growth. Here we hypothesized that the α-N-catenin, a homologous member of α-catenin and neural-specific expressed, functions as a novel tumor suppressor in neural crest-derived tumor, neuroblastoma. We correlated CTNNA2 (encodes α-N-catenin) expression to neuroblastoma disease relapse-free survival probability using publicly accessible human neuroblastoma datasets in R2 platform. The result showed that it negatively correlated to relapse-free survival probability significantly in patients with neuroblastoma with non-MYCN amplified tumor. Conversely, overexpressing CTNNA2 suppressed the neuroblastoma cell proliferation as measuring by the clonogenesis, inhibited anchorage-independent growth with soft agar colony formation assay. Forced expression of CTNNA2 decreased cell migration and invasion. Further, overexpression of CTNNA2 reduced the secretion of angiogenic factor IL-8 and HUVEC tubule formation. Our results show, for the first time, that α-N-catenin is a tumor suppressor in neuroblastoma cells. These findings were further corroborated with in vivo tumor xenograft study, in which α-N-catenin inhibited tumor growth and reduced tumor blood vessel formation. Interestingly, this is only observed in SK-N-AS xenografts lacking MYCN expression, and not in BE(2)-C xenografts with MYCN amplification. Mechanistically, α-N-catenin attenuated NF-κB responsive genes by inhibiting NF-κB transcriptional activity. In conclusion, these data demonstrate that α-N-catenin is a tumor suppressor in non-MYCN-amplified neuroblastomas and it inhibits NF-κB signaling pathway to suppress tumor growth in human neuroblastomas. Therefore, restoring the expression of α-N-catenin can be a novel therapeutic approach for neuroblastoma patients who have the deletion of CTNNA2 and lack of MYCN amplification.

Bromodomain and extraterminal inhibition blocks tumor progression and promotes differentiation in neuroblastoma.

BACKGROUND: MYCN amplification is a key molecular hallmark of high-risk neuroblastoma. Previously considered an "undruggable" target, MYCN transcription can be disrupted by inhibiting the bromodomain and the extraterminal (BET) domain family of proteins that regulates MYCN transcription epigenetically. JQ1 is a potent, small-molecule BET inhibitor that induces cell-cycle arrest and initiates apoptosis in neuroblastoma. Here, we sought to validate the antitumorigenic effects of JQ1 in neuroblastoma and to evaluate whether blocking N-myc expression with JQ1 promotes neural differentiation. METHODS: We determined the effects in vitro of JQ1 treatment on human neuroblastoma cell growth in both monolayer and sphere-forming conditions. Subcutaneous neuroblastoma xenografts were used for an in vivo study. Western blotting and immunohistochemistry were performed to evaluate the effects on neural differentiation and stem cell markers. RESULTS: JQ1 treatment blocked neuroblastoma cell growth in both monolayer and sphere-forming conditions; JQ1 also attenuated the growth of neuroblastoma xenograft in athymic nude mice. Neurofilament expression was enhanced with JQ1 treatment, indicating that JQ1 induces neuronal differentiation. Sphere forming conditions resulted in increased expression of multiple stem cell markers; these effects were reversed with JQ1 treatment. CONCLUSION: BET inhibition attenuates progression and promotes neural differentiation of neuroblastoma in vitro and in vivo in mice, providing insight into potential clinical applications of BET inhibitors in the treatment of patients with neuroblastoma.

NVP-BEZ-235 enhances radiosensitization via blockade of the PI3K/mTOR pathway in cisplatin-resistant non-small cell lung carcinoma.

INTRODUCTION: Most drug resistant cancer cells also develop resistance to radiation therapy. In this study, we hypothesized that the dual inhibitor of phosphatidylinositol-3 kinase/mammalian target of rapamycin, NVP-BEZ-235, could potentially enhance radiosensitization in cisplatin-resistance (CDDP-R) non-small cell lung cancer (NSCLC) cells by disabling autophagy as a mechanism of self-preservation. METHODS: We used both in vitro and in vivo approaches, including clonogenic assays, Western blotting, molecular analyses of autophagy and apoptosis, a xenograft model of tumor growth, and immunohistochemical analysis. RESULTS: Basal p-Akt, p-mTOR and p-S6R proteins were enhanced in CDDP-R NSCLC cells. CDDP-R-resistant NSCLC cells are less radiation sensitive in comparison to parental cells (DER=0.82, p=0.02); BEZ-235 enhanced the radiosensitivity (DER=1.2, p=0.01). In addition, combining BEZ-235/RT showed a dramatic tumor growth delay in a mouse xenograft model. Immunohistochemistry showed that combination therapy yielded 50% decrease in caspase-3 activity. Moreover, cell proliferation was reduced by 87.8% and vascular density by 86.1%. These results were associated with a downregulation of PI3K/mTOR signaling pathway and an increase in autophagy. CONCLUSIONS: These findings may be utilized as a novel strategy to enhance the efficacy of radiation therapy in drug-selected non-small cell lung cancer exhibiting radioresistance.

Targeting Aurora kinase-A downregulates cell proliferation and angiogenesis in neuroblastoma.

PURPOSE: Aurora kinase A (AURKA) overexpression is associated with poor prognosis in neuroblastoma and has been described to upregulate VEGF in gastric cancer cells. However, the exact role of AURKA in the regulation of neuroblastoma tumorigenesis remains unknown. We hypothesize that AURKA-mediated stabilization of N-Myc may affect VEGF expression and angiogenesis in neuroblastoma. Therefore, we sought to determine whether inhibition of AURKA modulates neuroblastoma angiogenesis. METHODS: Cell viability and anchorage-independent growth were determined after silencing AURKA or after treatment with MLN8237, AURKA inhibitor. Immunofluorescence was used to determine N-Myc localization. Human umbilical vein endothelial cells (HUVECs) were used to assess angiogenesis in vitro. Real time-PCR and ELISA were performed to determine VEGF transcription and secretion, respectively. RESULTS: Knockdown of AURKA significantly reduced cell proliferation and inhibited anchorage-independent growth. It also decreased N-Myc protein levels and nuclear localization. AURKA inhibition also decreased HUVECs tubule formation along with VEGF transcription and secretion. Similarly, MLN8237 treatment decreased neuroblastoma tumorigenicity in vitro. CONCLUSIONS: Our findings demonstrate that AURKA plays a critical role in neuroblastoma angiogenesis. AURKA regulates nuclear translocation of N-Myc in neuroblastoma cells, thus potentially affecting cell proliferation, anchorage-independent cell growth, and angiogenesis. Targeting AURKA might provide a novel therapeutic strategy in treating aggressive neuroblastomas.

Enhanced autophagy blocks angiogenesis via degradation of gastrin-releasing peptide in neuroblastoma cells.

Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.

Targeting gastrin-releasing peptide suppresses neuroblastoma progression via upregulation of PTEN signaling.

We have previously demonstrated the role of gastrin-releasing peptide (GRP) as an autocrine growth factor for neuroblastoma. Here, we report that GRP silencing regulates cell signaling involved in the invasion-metastasis cascade. Using a doxycycline inducible system, we demonstrate that GRP silencing decreased anchorage-independent growth, inhibited migration and neuroblastoma cell-mediated angiogenesis in vitro, and suppressed metastasis in vivo. Targeted inhibition of GRP decreased the mRNA levels of oncogenes responsible for neuroblastoma progression. We also identified PTEN/AKT signaling as a key mediator of the tumorigenic properties of GRP in neuroblastoma cells. Interestingly, PTEN overexpression decreased GRP-mediated migration and angiogenesis; a novel role for this, otherwise, understated tumor suppressor in neuroblastoma. Furthermore, activation of AKT (pAKT) positively correlated with neuroblastoma progression in an in vivo tumor-metastasis model. PTEN expression was slightly decreased in metastatic lesions. A similar phenomenon was observed in human neuroblastoma sections, where, early-stage localized tumors had a higher PTEN expression relative to pAKT; however, an inverse expression pattern was observed in liver lesions. Taken together, our results argue for a dual purpose of targeting GRP in neuroblastoma--1) decreasing expression of critical oncogenes involved in tumor progression, and 2) enhancing activation of tumor suppressor genes to treat aggressive, advanced-stage disease.

Autophagy mediates paracrine regulation of vascular endothelial cells.

Gastrin-releasing peptide (GRP) is a proangiogenic ligand secreted by tumors and acts directly upon binding to GRP receptor in endothelial cells. Angiogenesis plays a critical role in the pathology of various diseases, including cancer, as the formation of new blood vessels potentiates the rate of tumor growth and dissemination. GRP increases the migration of endothelial cells, but much is unknown about its role on endothelial cell proliferation and survival, as well as the signaling pathways involved. In the present study, we showed that GRP increases endothelial cell proliferation and tubule formation. There was a time-dependent increase in the levels of phosphorylated AKT, mammalian target of rapamycin (mTOR), and S6R in human umbilical vein endothelial cells treated with GRP. Interestingly, GRP treatment decreased the expression of proautophagic factors, ATG5, BECN1, and LC3 proteins. GRP also attenuated rapamycin-induced formation of autophagosomes. Moreover, overexpression of ATG5 or BECN1 significantly decreased tubule formation induced by exogenous GRP, whereas siRNA against ATG5 or BECN1 resulted in increased tubule formation with GRP treatment. Our results show that GRP inhibits the process of autophagy in vascular endothelial cells, thereby increasing endothelial cell proliferation and tubule formation. Here, we describe a novel role of GRP in the regulation of autophagy of endothelial cells, thereby providing a potential new therapeutic strategy in targeting angiogenesis during cancer progression.

Protein kinase C regulates bombesin-induced rapid VEGF secretion in neuroblastoma cells.

BACKGROUND: Intracellular signaling responsible for gastrin-releasing peptide (GRP) receptor-mediated neovascularization is not clearly understood. We sought to determine the cellular mechanisms involved in the GRP receptor regulation of vascular endothelial growth factor (VEGF) release in neuroblastoma cells. MATERIALS AND METHODS: BE(2)-C cells were treated with bombesin (BBS), the amphibian equivalent of GRP, Phorbol myristate acetate (PMA) a PKC agonist, or GF109293X (GFX), and analyses were performed for VEGF secretion, phosphorylated protein kinase B (AKT), extracellular signal-regulated kinases (ERK) and protein kinase D (PKD) expression. RESULTS: BBS rapidly increased VEGF secretion at 30 min. Pre-treatment with PMA alone produced similar results; this effect was synergistic with the addition of GRP. Conversely, GFX blocked PMA-stimulated increase in VEGF secretion. Immunofluorescent staining for VEGF correlated to BBS, PMA and GFX. CONCLUSION: PKC is critically responsible for rapid VEGF secretion by GRP receptor signaling in neuroblastoma cells. Inhibition of VEGF significantly reduced GRP-mediated cell proliferation, suggesting its crucial role in neuroblastoma tumorigenesis.

The zinc ionophore PCI-5002 radiosensitizes non-small cell lung cancer cells by enhancing autophagic cell death.

INTRODUCTION: A major focus of cancer research is to identify compounds that sensitize resistant cancer cells to radiation treatment. Lung cancer cells, in particular, have high rates of radioresistance that lead to treatment failure. We have previously shown that the autophagy induced in the context of decreased apoptosis confers radiosensitivity to prostate and lung cancer cells. Zinc supplementation has antiapoptotic effects in cell culture. In addition, the accumulation of zinc in response to oxidative stress has been associated with increased autophagy in astrocyte and breast cancer cells. METHODS: In this study, we hypothesized that the zinc ionophore PCI-5002 radiosensitizes lung cancer cells by inducing autophagic cell death. To test this hypothesis, we used a combination of in vitro and in vivo approaches, including clonogenic assays to test for radiosensitivity, biochemical analyses of apoptosis and autophagy, and a xenograft mouse model of tumor growth. RESULTS: We found that PCI-5002 reduced clonogenic survival in treated cells compared with untreated cells (0.03% versus 0.1% surviving fraction, p < 0.001). The increased radiosensitive fraction of PCI-5002-treated cells was accompanied by increased autophagy. PCI-5002 treatment also reduced caspase-3 cleavage. In an irradiated xenograft mouse model, the tumor growth of irradiated, PCI-5002-treated mice was slower than untreated, irradiated mice (25 days versus 22 days to reach a 1.0 cm tumor size). CONCLUSIONS: PCI-5002 treatment sensitizes lung cancer cells to radiation, both in vitro and in vivo. This data suggest that PCI-5002 could potentially treat radioresistant/locally advanced lung cancer by amplifying the effects of radiotherapy.

MLN8054, a small molecule inhibitor of aurora kinase a, sensitizes androgen-resistant prostate cancer to radiation.

PURPOSE: To determine whether MLN8054, an Aurora kinase A (Aurora-A) inhibitor causes radiosensitization in androgen-insensitive prostate cancer cells in vitro and in vivo. METHODS AND MATERIALS: In vitro studies consisted of culturing PC3 and DU145 prostate cancer cells and then immunoblotting Aurora A and phospho-Aurora A after radiation and/or nocodazole with MLN8054. Phases of the cell cycle were measured with flow cytometry. PC3 and DU145 cell lines were measured for survival after treatment with MLN8054 and radiation. Immunofluorescence measured γ-H2AX in the PC3 and DU145 cells after treatment. In vivo studies looked at growth delay of PC3 tumor cells in athymic nude mice. PC3 cells grew for 6 to 8 days in mice treated with radiation, MLN8054, or combined for 7 more days. Tumors were resected and fixed on paraffin and stained for von Willebrand factor, Ki67, and caspase-3. RESULTS: In vitro inhibition of Aurora-A by MLN8054 sensitized prostate cancer cells, as determined by dose enhancement ratios in clonogenic assays. These effects were associated with sustained DNA double-strand breaks, as evidenced by increased immunofluorescence for γ-H2AX and significant G2/M accumulation and polyploidy. In vivo, the addition of MLN8054 (30 mg/kg/day) to radiation in mouse prostate cancer xenografts (PC3 cells) significantly increased tumor growth delay and apoptosis (caspase-3 staining), with reduction in cell proliferation (Ki67 staining) and vascular density (von Willebrand factor staining). CONCLUSION: MLN8054, a novel small molecule Aurora-A inhibitor showed radiation sensitization in androgen-insensitive prostate cancer in vitro and in vivo. This warrants the clinical development of MLN8054 with radiation for prostate cancer patients.

Enhanced radiosensitivity of androgen-resistant prostate cancer: AZD1152-mediated Aurora kinase B inhibition.

Aurora kinase B (AURKB) is critical to the process of mitosis, aiding in chromosome condensation by phosphorylating histone H3. We investigated the effects of AZD1152, an AURKB inhibitor, on radiosensitivity of androgen-insensitive prostate cancer cells. The goal of this study was to test whether AZD1152 increases the susceptibility of hormone-refractory prostate cancer cells to radiation-induced DNA damage and to determine the conditions of AZD1152 treatment that maximize radiosensitization. PC3 and DU145 cells were treated with various AZD1152 doses for various durations to elucidate the conditions that yielded maximal increases in G(2)/M-phase and polyploid cells. To assess DNA damage, γ-H2AX phosphorylation was quantified for cells grown under radiosensitizing conditions and subjected to either no radiation or 5 Gy radiation. Radiosensitivity was determined by clonogenic assays. Cell cycle effects in both cell lines were maximized by treatment with 60 nM AZD1152 for 48 h. AZD1152-treated cells exhibited significantly increased DNA damage 30 min postirradiation (PC3: 100% compared to 68%, P  =  0.035; DU145: 100% compared to 69%, P  =  0.034), with additional DNA damage 6 h postirradiation (PC3: 85% compared to 15%, P  =  0.002; DU145: 67% compared to 21%, P  =  0.012). Radiosensitivity was increased in both cell lines, with dose enhancement ratios of 1.53 for PC3 cells (P  =  0.017) and 1.71 for DU145 cells (P  =  0.02). This study identifies the optimal AZD1152 treatment conditions to maximize the radiosensitization of PC3 and DU145 cells. These results suggest a major role for DNA damage and impairment of DNA repair mechanisms in AZD1152-induced radiosensitization of prostate cancer cells.

AT-101, a pan-Bcl-2 inhibitor, leads to radiosensitization of non-small cell lung cancer.

INTRODUCTION: Radiotherapy has a central role in the treatment of lung cancer. However, its effectiveness is often limited, in part, because of the defects in key apoptosis regulators, such as Bcl-2 family members, that contribute to cancer ability to evade apoptosis. In this study, we tested AT-101, a pan-Bcl-2 inhibitor, as a potential radiosensitizer in lung cancer. METHODS AND RESULTS: Clonogenic assays were used to determine the radiosensitivity of multiple lung cancer cell lines. On the basis of their relative response to radiotherapy, lung cancer cells were stratified into two groups, and a representative cell line of each group was selected for more in-depth study: A549 (resistant) and HCC2429 (sensitive). The expression levels of antiapoptotic (Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic (Bax, Bak, and Bid) Bcl-2 proteins were determined for each group. Although the levels of Bcl-2 and Mcl-1 were low for both groups, Bcl-XL expression was dramatically higher in A549, whereas almost not detected in HCC2429. The levels of Bax/Bak were 40% higher in HCC2429 compared with A549. When administered alone, AT-101 resulted in increased apoptosis in concentration-dependent manner against both groups, with enhanced activity in HCC2429 even at lower concentration. Furthermore, AT-101 promoted radiosensitivity of A549 and HCC2429 cells (p < 0.005). Consistent with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay findings, A549 cells required increased AT-101 dose to achieve a similar cytoxicity to HCC2429 cells. CONCLUSIONS: These investigations suggest that the Bcl-2 family may serve as effective therapeutic targets in lung cancer. Further clinical studies are warranted to assess the potential of AT-101 as an agent that enhances the therapeutic ratio of radiotherapy in lung cancer.

Enzastaurin (LY317615), a protein kinase C beta selective inhibitor, enhances antiangiogenic effect of radiation.

PURPOSE: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. METHODS AND MATERIALS: The combination of the selective PKCbeta inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. RESULTS: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. CONCLUSION: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

Combined Bcl-2/mammalian target of rapamycin inhibition leads to enhanced radiosensitization via induction of apoptosis and autophagy in non-small cell lung tumor xenograft model.

PURPOSE: Radiotherapy has a central role in the treatment of non-small cell lung cancer. Effectiveness of this modality, however, is often limited as resistance results from defects in cell death. EXPERIMENTAL DESIGN: We investigated whether simultaneous up-regulation of apoptosis, via Bcl-2 inhibitor ABT-737, and autophagy, via mammalian target of rapamycin inhibitor rapamycin, can be used to enhance radiosensitivity of H460 cells in vitro and growth delay in a xenograft model. RESULTS: In vitro studies confirmed that ABT-737 and rapamycin induce apoptosis and autophagy, respectively. ABT-737 induced cleaved caspase-3, a marker of apoptosis, and rapamycin correlated with an increase in punctate localization of green fluorescent protein-LC3, characteristic of autophagy. The combination ABT-737/rapamycin markedly enhanced sensitivity of H460 cells to radiation (dose enhancement ratio = 2.47; P = 0.002) in clonogenic assay. In addition, the combination ABT-737/rapamycin/radiation showed a dramatic tumor growth delay in a mouse xenograft model. In vivo immunohistochemistry staining showed that combination therapy yielded over a 100% increase in caspase-3 activity (apoptosis) and a 6-fold decrease in p62 protein level (indicative of autophagic flux) compared with radiation alone control group. Moreover, cell proliferation (Ki-67 staining) was reduced by 77% (P = 0.001) and vascular density (von Willebrand factor staining) by 67.5% (P = 0.09) compared with radiation alone. Additional in vitro studies in human umbilical vein endothelial cells indicated that combined therapy also significantly decreases tubule formation. CONCLUSION: These results suggest that concurrent induction of apoptosis and autophagy enhances radiation therapy both in vitro and in lung cancer xenograft models. Further investigations are warranted to assess the clinical potential of such strategy in lung cancer patients.