Seyedmehrad Tavallai

The role of cell signalling in the crosstalk between autophagy and apoptosis

Not surprisingly, the death of a cell is a complex and well controlled process. For several decades, apoptosis, the first genetically programmed death process to be identified has taken centre stage as the principal mechanism of programmed cell death (type I cell death) in mammalian tissues. Apoptosis has been extensively studied and its contribution to the pathogenesis of disease well documented. However, apoptosis does not function alone in determining the fate of a cell. More recently, autophagy, a process in which de novo formed membrane enclosed vesicles engulf and consume cellular components, has been shown to engage in complex interplay with apoptosis. As a result, cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II cell death is not completely clear and as we will discuss in this review and perhaps a discrete difference does not exist, due to intrinsic factors among different cell types and crosstalk among organelles within each cell type. Apoptosis may begin with autophagy and autophagy can often end with apoptosis, inhibition or a blockade of caspase activity may lead a cell to default into Type II cell death from Type I.

GRP78/BiP/HSPA5/Dna K is a Universal Therapeutic Target for Human Disease

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU‐03012 (AR‐12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU‐03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre‐treatment with OSU‐03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU‐03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi‐drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU‐03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU‐03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. J. Cell. Physiol. 230: 1661–1676, 2015.

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.

PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L‐NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over‐expression of c‐FLIP‐s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. J. Cell. Physiol. 230: 1115–1127, 2015.

Sorafenib/regorafenib and lapatinib interact to kill CNS tumor cells.

The present studies were to determine whether the multi‐kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3‐GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c‐FLIP‐s, 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 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy. J. Cell. Physiol. 230: 131–139, 2015.

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.

Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy

The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.

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.

Abstract 2690: Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The present study aimed to determine whether the multikinase inhibitors, sorafenib/regorafenib, worked in conjunction with PI3K/AKT inhibitors to enhance tumor cell death. It has been noted that combination of sorafenib/regorafenib with a PI3K inhibitor acetic acid (PX-866) resulted in tumor cell death, in a greater than additive fashion, in liver, colorectal, lung, breast, kidney and brain cancer cells. Similar data was obtained using AKT inhibitors perifosine and MK2206. Furthermore, even cells lacking PTEN remained as sensitive to this combinational approach as cells expressing PTEN. PX-866 treatment abolished AKT/GSK3 phosphorylation, with tumor cell death correlating with reduced activity of AKT and 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-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, LAMP2 and LC3/LC3II which correlated with the resulting increase in LC3-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 ATG5 suppressed drug toxicity by ∼40%. In vivo, combination of either sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrates that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo. Citation Format: Larry A. Booth, Nichola A. Cruickshanks, G B. Sajithlal, Hossein A. Hamed, Seyedmehrad Tavallai, J Syed, Steven Grant, Andrew Poklepovic, Paul Dent. Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2014-2690

Abstract 796: Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Detachment of cells from the extracellular matrix (ECM) results in a form of cell death referred to as anoikis. Anoikis resistance (AR), frequently acquired by malignant cells, is required for tumor metastases. The present study focused on identifying the mechanisms by which drug sensitivity is restored in previously resistant AR breast cancer cells. AR breast cancer cells display reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA and PUMA. In contrast, expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. It has previously been noted that AR cells display resistance to several currently available anti-tumor cell therapies, including a combinational approach with Lapatinib (ERBB1/2 inhibitor) and Obatoclax (MCL-1 inhibitor, exhibiting reduced autophagic flux, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL in AR cells resulted in apoptosis to a greater extent than in their wild-type counterparts. Pre-treatment of AR cells with Valproate (histone deacetylase inhibitor (HDACIs)); for 24hrs increased the levels of toxic BH3 domain proteins whilst simultaneously reducing levels of MCL-1 and re-sensitising AR cells to therapy-mediated cell death. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored chemo-sensitivity and prolonged animal survival. These data argue that pre-treatment with HDACIs may provide a mechanism to enhance the anti-tumor effect of chemotherapy in AR breast cancer cells. Citation Format: Nichola A. Cruickshanks, Hossein A. Hamed, Larry A. Booth, Seyedmehrad Tavallai, G B. Sajithlal, Steven Grant, Andrew Poklepovic, Paul Dent. Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2014-796