The CHK1 inhibitor SRA737 synergizes with PARP1 inhibitors to kill carcinoma cells

Inhibitors of PARP1 are approved therapeutic agents in ovarian carcinomas. We determined whether the novel clinically relevant CHK1 inhibitor SRA737 interacted with PARP1 inhibitors to kill carcinoma cells. In multiple mammary and ovarian cancer lines SRA737 synergized with the PARP1 inhibitors olaparib and niraparib to cause cell death. The [SRA737 + niraparib] drug combination activated an ATM-AMPK-ULK1-mTOR pathway which resulted in the formation of autophagosomes, temporally followed by autolysosome formation. Phosphorylation of ULK1 S317 was essential for kinase activation against ATG13. The drug combination elevated eIF2α phosphorylation which was causal at increasing Beclin1 and ATG5 expression, reducing MCL-1 and BCL-XL levels, and causing CD95 activation. Knock down of CD95, eIF2α, ATM, AMPKα, ULK1, Beclin1 or ATG5 reduced drug combination lethality. Blockade of either caspase 9 function or that of AIF each partially prevented cell death. Expression of activated mTOR or of c-FLIP-s or of BCL-XL reduced cell killing. In vivo, SRA737 and niraparib interacted in an additive fashion to suppress the growth of mammary tumors. Multiplex analyses revealed that drug combination treated tumors had reduced their plasma levels of sERBB1, sERBB2, sVEGFR1, sVEGFR2, sIL-6R, HGF, PDGFAB/BB and CXCL16 and enhanced the levels of CCL26, IL-8 and MIF. Surviving tumors had activated ERK1/2 and AKT. This finding argues that IL-8/ERK/AKT signaling may be an evolutionary survival response to [SRA737 + niraparib].     

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the PARP1 inhibitor niraparib to kill ovarian cancer cells

The irreversible ERBB1/2/4 inhibitor neratinib has been shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET, PDGFRα and mutant RAS proteins via autophagic degradation. Neratinib interacted in an additive to synergistic fashion with the approved PARP1 inhibitor niraparib to kill ovarian cancer cells. Neratinib and niraparib caused the ATM-dependent activation of AMPK which in turn was required to cause mTOR inactivation, ULK-1 activation and ATG13 phosphorylation. The drug combination initially increased autophagosome levels followed later by autolysosome levels. Preventing autophagosome formation by expressing activated mTOR or knocking down of Beclin1, or knock down of the autolysosome protein cathepsin B, reduced drug combination lethality. The drug combination caused an endoplasmic reticulum stress response as judged by enhanced eIF2α phosphorylation that was responsible for reducing MCL-1 and BCL-XL levels and increasing ATG5 and Beclin1 expression. Knock down of BIM, but not of BAX or BAK, reduced cell killing. Expression of activated MEK1 prevented the drug combination increasing BIM expression and reduced cell killing. Downstream of the mitochondrion, drug lethality was partially reduced by knock down of AIF, but expression of dominant negative caspase 9 was not protective. Our data demonstrate that neratinib and niraparib interact to kill ovarian cancer cells through convergent DNA damage and endoplasmic reticulum stress signaling. Cell killing required the induction of autophagy and was cathepsin B and AIF -dependent, and effector caspase independent.     

Pazopanib and HDAC inhibitors interact to kill sarcoma cells

The present studies were to determine whether the multi-kinase inhibitor pazopanib interacted with histone deacetylase inhibitors (HDACI: valproate, vorinostat) to kill sarcoma cells. In multiple sarcoma cell lines, at clinically achievable doses, pazopanib and HDACI interacted in an additive to greater than additive fashion to cause tumor cell death. The drug combination increased the numbers of LC3-GFP and LC3-RFP vesicles. Knockdown of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, and to a lesser extent BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 was required to strongly suppress drug combination lethality. The drug combination inactivated mTOR and expression of activated mTOR strongly suppressed drug combination lethality. Treatment of animals carrying sarcoma tumors with pazopanib and valproate resulted in a greater than additive reduction in tumor volume compared with either drug individually. As both pazopanib and HDACIs are FDA-approved agents, our data argue for further determination as to whether this drug combination is a useful sarcoma therapy in the clinic.     

Palbociclib augments Neratinib killing of tumor cells that is further enhanced by HDAC inhibition

Cancers expressing mutant RAS are associated with a weaker response to chemotherapy and a shorter overall patient survival. We have demonstrated that the irreversible inhibitor of ERBB1/2/4, neratinib, inhibits ERBB1/2/4 and causes their internalization and autolysosomal degradation. Fellow-traveler membrane proteins with RTKs, including mutant K-/N-RAS, were also degraded. We discovered that the CDK4/6 inhibitor palbociclib increased autophagosome and then autolysosome levels in a time dependent fashion, did not reduce mTOR activity, and interacted with temsirolimus to kill. Neratinib and palbociclib interacted in a greater than additive manner to increase autophagosome and then autolysosome levels in a time dependent fashion, and to cause tumor cell killing. Killing required the expression of ATM and AMPKα, Beclin1 and ATG5, BAX and BAK and of AIF, but not of caspase 9. In some cells over-expression of BCL-XL was protective whereas in others it was ineffective. The lethality of [neratinib + palbociclib] was modestly enhanced by the PDE5 inhibitor sildenafil and strongly enhanced by the HDAC inhibitor sodium valproate. This was associated with K-RAS degradation and a greater than additive increase in autophagosome and autolysosome levels. Killing by the three-drug combination required ATM and AMPKα, and, to a greater extent, Beclin1 and ATG5. In vivo, [valproate + palbociclib] and [neratinib + valproate + palbociclib] interacted to suppress the growth of a carboplatin/paclitaxel resistant PDX ovarian tumors that express a mutant N-RAS. Our data support performing a future three-drug trial with these agents.     

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells

The irreversible ERBB1/2/4 inhibitor, neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of neratinib to promote greater levels of cell killing.     

PARP inhibitors in older patients with ovarian and breast cancer: Young International Society of Geriatric Oncology review paper

Breast and ovarian cancer are common malignancies among older adults, causing significant morbidity and mortality. Although most cases of breast and ovarian cancer are sporadic, a significant proportion is caused by mutations in cancer susceptibility genes, most often breast cancer susceptibility genes (BRCA) 1 and 2. Furthermore, some breast and ovarian tumors are phenotypically similar to those with BRCA mutations, a phenomenon known as “BRCAness”. BRCA mutations and “BRCAness” lead to defects in DNA repair, which may be a target for therapeutic agents such as Poly ADP-Ribose Polymerase (PARP) inhibitors. PARP inhibitors are novel medications which lead to double-strand breaks resulting in cell death due to synthetic lethality, and which have been shown to be effective in patients with advanced breast and ovarian cancers with or without BRCA mutations. Three different PARP inhibitors (olaparib, niraparib, and rucaparib) have been approved for the treatment of ovarian cancer and one (olaparib) for breast cancer harboring BRCA mutations. Here, we review the currently available evidence regarding the use of PARP inhibitors for the treatment of patients with breast and ovarian cancer, with a particular focus on the inclusion of older adults in clinical trials of these therapies. Additionally, we provide an overview of currently ongoing studies of PARP inhibitors in breast and ovarian cancer, and include recommendations for increasing the evidence-base for using these medications among older patients.     

Regulation of dimethyl-fumarate toxicity by proteasome inhibitors

The present studies examined the biology of the multiple sclerosis drug dimethyl-fumarate (DMF) or its in vivo breakdown product and active metabolite mono-methyl-fumarate (MMF), alone or in combination with proteasome inhibitors, in primary human glioblastoma (GBM) cells. MMF enhanced velcade and carfilzomib toxicity in multiple primary GBM isolates. Similar data were obtained in breast and colon cancer cells. MMF reduced the invasiveness of GBM cells, and enhanced the toxicity of ionizing radiation and temozolomide. MMF killed freshly isolated activated microglia which was associated with reduced IL-6, TGFβ and TNFα production. The combination of MMF and the multiple sclerosis drug Gilenya further reduced both GBM and activated microglia viability and cytokine production. Over-expression of c-FLIP-s or BCL^-XL protected GBM cells from MMF and velcade toxicity. MMF and velcade increased plasma membrane localization of CD95, and knock down of CD95 or FADD blocked the drug interaction. The drug combination inactivated AKT, ERK1/2 and mTOR. Molecular inhibition of AKT/ERK/mTOR signaling enhanced drug combination toxicity whereas molecular activation of these pathways suppressed killing. MMF and velcade increased the levels of autophagosomes and autolysosomes and knock down of ATG5 or Beclin1 protected cells. Inhibition of the eIF2α/ATF4 arm or the IRE1α/XBP1 arm of the ER stress response enhanced drug combination lethality. This was associated with greater production of reactive oxygen species and quenching of ROS suppressed cell killing.     

Sorafenib and HDAC inhibitors synergize to kill CNS tumor cells

The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.     

Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation

We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2alpha phosphorylation; suppression of eIF2alpha function abolished drug combination lethality. Cell killing was paralleled by PERK-and eIF2alpha-dependent lowering of c-FLIP-s protein levels and overexpression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRbeta and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Overexpression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2alpha- downward arrowc-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).     

PDE5 inhibitors enhance the lethality of standard of care chemotherapy in pediatric CNS tumor cells

We determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill medulloblastoma cells. In medulloblastoma cells PDE5 inhibitors interacted in a greater than additive fashion with vincristine/etoposide/cisplatin to cause cell death. Knockdown of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of dominant negative caspase 9 did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with the PDE5 inhibitor sildenafil. Overexpression of BCL-XL and c-FLIP-s suppressed individual and combination drug toxicities. Knockdown of CD95 or FADD suppressed drug combination toxicity. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy which was maximal at ~12 h post-treatment, and in a cell type-dependent manner knockdown of Beclin1 or ATG5 either suppressed or enhanced drug combination lethality. PDE5 inhibitors enhanced the induction of chemotherapy-induced DNA damage in a nitric oxide synthase-dependent fashion. In conclusion, our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for medulloblastoma represents a possible novel modality for future treatment of this disease.     

A Prospective Phase II Single-arm Study of Niraparib Plus Dostarlimab in Patients With Advanced Non–small-cell Lung Cancer and/or Malignant Pleural Mesothelioma,Positive for PD-L1 Expression and Germline or Somatic Mutations in the DNA Repair Genes: Rationale and Study Design

Treatment with poly ADP ribose polymerase (PARP)1/2 inhibitors represents a novel opportunity to selectively kill a subset of cancer cell types by exploiting their deficiencies in DNA repair, thus leading to synthetic lethality. Treatment of homologous recombination deficient (HRD)-tumors with PARP inhibitors generates significant levels of DNA damage, which has the potential to further increasing tumor mutational burden, promoting neoantigen release, and upregulating both interferons and programmed death ligand-1 (PD-L1) expression, suggesting a potential complementary and synergistic role with immune checkpoint inhibitors in cancer treatment. Here we present the design and rationale of a prospective, phase II, single-arm study aiming to investigate the safety and antitumor activity of the combination of niraparib and dostarlimab in patients with HRD-positive and PD-L1 ≥ 1% advanced non–small-cell lung cancer (NSCLC) and/or malignant pleural mesothelioma (MPM).Considering the prevalence of pathogenetic germline mutations in DNA repair genes, reported to be around 5% to 10% in patients with MPM and NSCLC, a total of 700 to 1000 cases will be screened to identify 70 patients who are HRD-positive/PD-L1 ≥ 1% (N = 35 NSCLC; N = 35 MPM) to be included. Patients will receive the combination of niraparib orally once daily and dostarlimab intravenously. The primary endpoint is progression-free survival. Secondary endpoints are objective response, duration of response, overall survival, and safety.The results of this study will provide evidence on the safety and antitumor activity of niraparib and dostarlimab combination in patients with advanced, HRD-positive and PD-L1 ≥ 1% NSCLC and/or MPM.     

HDAC inhibitors enhance the lethality of low dose salinomycin in parental and stem-like GBM cells

The present studies determined whether the antibiotic salinomycin interacted with HDAC inhibitors to kill primary human GBM cells. Regardless of PTEN, ERBB1, or p53 mutational status salinomycin interacted with HDAC inhibitors in a synergistic fashion to kill GBM cells. Inhibition of CD95/Caspase 8 or of CD95/RIP-1/AIF signaling suppressed killing by the drug combination. Salinomycin increased the levels of autophagosomes that correlated with increased p62 and LC3II levels; valproate co-treatment correlated with reduced LC3II and p62 expression, and increased caspase 3 cleavage. Molecular inhibition of autophagosome formation was protective against drug exposure. The drug combination enhanced eIF2α phosphorylation and decreased expression of MCL-1 and phosphorylation of mTOR and p70 S6K. Activation of p70 S6K or mTOR promoted cell survival in the face of combined drug exposure. Overexpression of BCL-XL or c-FLIP-s was protective. Collectively our data demonstrate that the lethality of low nanomolar concentrations of salinomycin are enhanced by HDAC inhibitors in GBM cells and that increased death receptor signaling together with reduced mitochondrial function are causal in the combinatorial drug necro-apoptotic killing effect.     

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.     

Histone deacetylase inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis in human malignant glioma cells

Histone deacetylase (HDAC) inhibitors have both apoptotic and differentiating effects on various tumor cells. However, the mechanisms underlying the effect of HDAC inhibitors remain unclear. In this study, we investigated the function of anti-proliferative effects of HDAC inhibitors, N-butyric acid and trichostatin A, on human malignant glioma cell lines, U251-MG and D54. MTT assay showed a dose-dependent inhibition of cellular proliferation in both cell lines. Cell cycle analysis revealed increased sub-G1 population in both lines, and G1 arrest only in U251-MG cells. Induction of apoptosis was also supported by the occurrence of DNA fragmentation in tumor cells treated with HDAC inhibitors. Furthermore, caspase inhibition assay indicated that HDAC inhibitor-induced apoptosis was caspase-dependent. Neither mitochondrial membrane potential nor the expression of caspase-9 was changed by treatment with HDAC inhibitors, suggesting the possibility that HDAC inhibitor-induced apoptosis was not mediated by the mitochondrial cell death pathway. On the other hand, immunoblot assay confirmed increased expression of caspase-8 in both lines, and elevation of p21 but not p27 protein in U251-MG cells following HDAC inhibitor treatment. Taken together, the HDAC inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis with or without p21-mediated G1 arrest in human malignant glioma cells.     

Fingolimod augments Pemetrexed killing of non-small cell lung cancer and overcomes resistance to ERBB inhibition

Overall, NSCLC has a poor 5-year survival and new therapeutic approaches are urgently needed. ERBB-addicted NSCLC that have become resistant to ERBB inhibitors are often refractory to additional therapeutic interventions. The sphingosine-1-phosphate receptor modulator fingolimod (FTY720), approved for the treatment of multiple sclerosis, synergized with the NSCLC therapeutic pemetrexed to kill NSCLC and ovarian cancer cells. This occurred in lung cancer cells expressing mutated K-RAS, mutated ERBB1, or in NSCLC cells resistant to afatinib (an ERBB1/2/4 inhibitor). This drug combination appeared to use overlapping and distinct mechanisms of killing in different cell lines. Activation of AMP-dependent kinase (AMPK) and reduced expression and inactivation of mTOR were associated with increased autophagosome and autolysosome formation. Downregulation of Beclin1 considerably reduced formation of autophagosomes and protected the cells from drug combination-induced killing without significantly altering autolysosome formation. Autophagy protein 5 (ATG5) knock down afforded greater protection against the combination of pemetrexed with fingolimod. Treatment of cells with the mTOR inhibitor everolimus markedly enhanced the lethality of pemetrexed plus fingolimod combination. Our data suggest that the combination of fingolimod with the established NSCLC/ovarian cancer drug pemetrexed should be explored as a new therapy.     

Niraparib for the treatment of ovarian cancer

Introduction: Niraparib, an orally available selective inhibitor of poly(adenosine diphosphate-ribose) polymerase (PARP), is the first PARP inhibitor approved for use in patients with ovarian cancer who do not harbor a germ-line or somatic mutation in the breast cancer gene (BRCA). Overall, niraparib is well tolerated and its toxicities, primarily hematologic, are manageable especially with recently released initial dose modification guidelines based on weight and baseline platelet count. The role of niraparib as maintenance following frontline platinum-based chemotherapy as well as in the treatment of recurrent high-grade serous ovarian cancer is an active area of investigation.

Areas covered: This review focuses on niraparib, its pharmacology, clinical efficacy, and adverse effects as evidenced by prospective clinical trials, and licensed indications.

Expert commentary: Niraparib introduced the use of PARP inhibitors regardless of biomarker status. Recent studies highlight the critical need for more accurate biomarkers to identify patients most likely to benefit from treatment with PARP inhibitors. In the next 5 years, we anticipate further expansion of and elucidation regarding the optimal indication for use of niraparib in the treatment of ovarian cancer.     

[pemetrexed + sildenafil], via autophagy-dependent HDAC downregulation,enhances the immunotherapy response of NSCLC cells

Pemetrexed is an approved therapeutic in NSCLC and ovarian cancer. Our studies focused on the ability of [pemetrexed + sildenafil] exposure to alter the immunogenicity of lung and ovarian cancer cells. Treatment of lung and ovarian cancer cells with [pemetrexed + sildenafil] in vitro rapidly reduced the expression of PD-L1, PD-L2 and ornithine decarboxylase (ODC), and increased the expression of class I MHCA. In a cell-specific fashion, some cells also released the immunogenic nuclear protein HMGB1 into the extracellular environment. [Pemetrexed + sildenafil] reduced the expression of multiple histone deacetylases that was blocked by knock down of autophagy regulatory proteins. [Pemetrexed + sildenafil] lethality was enhanced by the histone deacetylase inhibitors AR42 and sodium valproate; AR42 and valproate as single agents also rapidly reduced the expression of PD-L1, PD-L2 and ODC, and increased expression of MHCA and CerS6. Nitric oxide and CerS6 signaling was required for drug-induced death receptor activation and tumor cell killing. In vivo, [pemetrexed + sildenafil] lethality against lung cancer cells was enhanced by sodium valproate. Using syngeneic mouse lung cancer cells [pemetrexed + sildenafil] enhanced the anti-tumor effects of antibodies directed to inhibit PD-1 or CTLA4. [Pemetrexed + sildenafil] interacted with the anti-PD-1 antibody to strongly enhance tumor infiltration by M1 macrophages; activated NK cells and activated T cells. Our data demonstrate that treatment of tumor cells with [pemetrexed + sildenafil] results in tumor cell killing and via autophagy-dependent downregulation of HDACs, it opsonizes the remaining tumor cells to anti-tumor immunotherapy antibodies.     

Chromatin remodeling underlies the senescence-associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression

Age is a major risk factor for the development of cancer. Senescent fibroblasts, which accumulate with age, secrete protumorigenic factors collectively referred to as the senescence-associated secretory phenotype (SASP). Here, we examined the molecular mechanisms that control SASP activation, focusing on the known SASP factor osteopontin (OPN). We found that expression of the canonical SASP members interleukin (IL)-6 and IL-8, but not OPN, were dependent upon a persistent DNA damage response (DDR) as evidenced by ATM and NF-κB activation. Treatment with several histone deacetylase (HDAC) inhibitors robustly activated SASP in the absence of DNA breaks, suggesting that DDR-dependent SASP activation occurs in response to chromatin remodeling rather than physical breaks in DNA. In the setting of HDAC inhibition, IL-6 and IL-8 expression remained dependent upon ATM and NF-κB, while OPN expression remained independent of these factors. Further analysis revealed that HDAC1 inhibition was sufficient to induce OPN expression, which is interesting given that loss of HDAC1 expression correlates with increased OPN expression within the stromal compartment of invasive breast cancers. Importantly, fibroblasts treated with HDAC inhibitors promoted tumor growth in vivo. Our findings therefore indicate that HDAC modulation plays an important role in stromal cell activation, with important implications for the use of HDAC inhibitors in the treatment of cancer.     

IL-13 regulates cancer invasion and metastasis through IL-13Rα2 via ERK/AP-1 pathway in mouse model of human ovarian cancer

Previously, we have demonstrated that a variety of human cancers including the ovarian cancer express IL-13Rα2, a high affinity receptor for IL-13. Herein, we have examined if IL-13 regulates invasion and metastasis of ovarian cancer through IL-13Rα2 in vitro and in vivo in animal models of human ovarian cancer. We tested cell invasion and protease activity in IL-13Rα2-overexpressing and IL-13Rα2-negative ovarian tumor cell lines. IL-13 treatment significantly augmented both cell invasion and enzyme activities in only IL-13Rα2-positive cells but not in IL-13Rα2-negative cells in vitro. Mechanistically, IL-13 enhanced ERK1/2, AP-1 and MMP activities only in IL-13Rα2-positive cells but not in IL-13Rα2-negative cells. In contrast, other signaling pathways such as IRS1/2, PI3K and AKT do not seem to be involved in IL-13 induced signaling in ovarian cancer cell lines. Highly specific inhibitors for MMP and AP-1 efficiently inhibited both invasion and protease activities without impacting the basal level invasion and protease activities in vitro. In orthotopic animal model of human ovarian cancer, IL-13Rα2-positive tumors metastasized to lymph nodes and peritoneum earlier than IL-13Rα2-negative tumors. Interestingly, the IL-13Rα2-positive tumor bearing mice died earlier than mice with IL-13Rα2-negative tumor. Intraperitoneal injection of IL-13 further shortened survival of IL-13Rα2-positive tumor bearing mice compared to IL-13Rα2-negative tumor mice. IL-13Rα2-positive tumors and lymph node metastasis expressed higher levels of MMPs and higher ERK1/2 activation compared to IL-13Rα2-negative tumors. Taken together, IL-13Rα2 is involved in cancer metastasis through activation of ERK/AP-1 and that targeting IL-13Rα2 might not only directly kill primary tumors but also prevent cancer metastasis.