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Glioma stem cells (GSCs) comprise a small subpopulation of glioblastoma multiforme cells that contribute to therapy resistance, poor prognosis, and tumor recurrence. Protective autophagy promotes resistance of GSCs to anoikis, a form of programmed cell death occurring when anchorage-dependent cells detach from the extracellular matrix. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of melanoma differentiation associated gene-9/Syntenin (MDA-9) (syndecan binding protein; SDCBP). When MDA-9 is suppressed, GSCs undergo autophagic death supporting the hypothesis that MDA-9 regulates protective autophagy in GSCs under anoikis conditions. MDA-9 maintains protective autophagy through phosphorylation of BCL2 and by suppressing high levels of autophagy through EGFR signaling. MDA-9 promotes these changes by modifying FAK and PKC signaling. Gain-of-function and loss-of-function genetic approaches demonstrate that MDA-9 regulates pEGFR and pBCL2 expression through FAK and pPKC. EGFR signaling inhibits autophagy markers (ATG5, Lamp1, LC3B), helping to maintain protective autophagy, and along with pBCL2 maintain survival of GSCs. In the absence of MDA-9, this protective mechanism is deregulated; EGFR no longer maintains protective autophagy, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. In addition, pBCL2 is down-regulated in the absence of MDA-9, leading to cell death in GSCs under conditions of anoikis. Our studies confirm a functional link between MDA-9 expression and protective autophagy in GSCs and show that inhibition of MDA-9 reverses protective autophagy and induces anoikis and cell death in GSCs.

Apoptosis and autophagy play seminal roles in maintaining organ homeostasis. Apoptosis represents canonical type I programmed cell death. Autophagy is viewed as pro-survival, however, excessive autophagy can promote type II cell death. Defective regulation of these two obligatory cellular pathways is linked to various diseases, including cancer. Biologic or chemotherapeutic agents, which can reprogram cancer cells to undergo apoptosis- or toxic autophagy-mediated cell death, are considered effective tools for treating cancer. Melanoma differentiation associated gene-7 (mda-7) selectively promotes these effects in cancer cells. mda-7 was identified more than two decades ago by subtraction hybridization showing elevated expression during induction of terminal differentiation of metastatic melanoma cells following treatment with recombinant fibroblast interferon and mezerein (a PKC activating agent). MDA-7 was classified as a member of the IL-10 gene family based on its chromosomal location, and the presence of an IL-10 signature motif and a secretory sequence, and re-named interleukin-24 (MDA-7/IL-24). Multiple studies have established MDA-7/IL-24 as a potent anti-cancer agent, which when administered at supra-physiological levels induces growth arrest and cell death through apoptosis and toxic autophagy in a wide variety of tumor cell types, but not in corresponding normal/non-transformed cells. Furthermore, in a phase I/II clinical trial, MDA-7/IL-24 administered by means of a non-replicating adenovirus was well tolerated and displayed significant clinical activity in patients with multiple advanced cancers. This review examines our current comprehension of the role of MDA-7/IL-24 in mediating cancer-specific cell death via apoptosis and toxic autophagy. [ABSTRACT FROM AUTHOR]

X-linked inhibitor of apoptosis protein (XIAP) is constitutively expressed endogenous inhibitor of apoptosis, exhibit its antiapoptotic effect by inactivating key caspases such as caspase-3, caspase-7 and caspase-9 and also play pivotal role in rendering cancer chemoresistance. Our studies showed the coadministration of TQ and TAM resulting in a substantial increase in breast cancer cell apoptosis and marked inhibition of cell growth both in vitro and in vivo. Anti-angiogenic and anti-invasive potential of TQ and TAM was assessed through in vitro studies. This novel combinatorial regimen leads to regulation of multiple cell signaling targets including inactivation of Akt and XIAP degradation. At molecular level, TQ and TAM synergistically lowers XIAP expression resulting in binding and activation of caspase-9 in apoptotic cascade, and interfere with cell survival through PI3-K/Akt pathway by inhibiting Akt phosphorylation. Cleaved caspase-9 further processes other intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis, indicated by rise in the sub-G1 cell population. This combination also downregulates the expression of Akt-regulated downstream effectors such as Bcl-xL, Bcl-2 and induce expression of Bax, AIF, cytochrome C and p-27. Consistent with these results, overexpression studies further confirmed the involvement of XIAP and its regulatory action on Akt phosphorylation along with procaspase-9 and PARP cleavage in TQ-TAM coadministrated induced apoptosis. The ability of TQ and TAM in inhibiting XIAP was confirmed through siRNA-XIAP cotransfection studies. This novel modality may be a promising tool in breast cancer treatment. [ABSTRACT FROM AUTHOR]

Melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24), a secreted protein of the IL-10 family, was first identified more than two decades ago as a novel gene differentially expressed in terminally differentiating human metastatic melanoma cells. MDA-7/IL-24 functions as a potent tumor suppressor exerting a diverse array of functions including the inhibition of tumor growth, invasion, angiogenesis, and metastasis, and induction of potent "bystander" antitumor activity and synergy with conventional cancer therapeutics. MDA-7/IL-24 induces cancer-specific cell death through apoptosis or toxic autophagy, which was initially established in vitro and in preclinical animal models in vivo and later in a Phase I clinical trial in patients with advanced cancers. This review summarizes the history and our current understanding of the molecular/biological mechanisms of MDA-7/IL-24 action rendering it a potent cancer suppressor. [ABSTRACT FROM AUTHOR]

Progression Elevated Gene-3 (PEG-3) was cloned using subtraction hybridization as an upregulated transcript associated with transformation and tumor progression of rat embryo fibroblast cells. PEG-3 is a unique gene facilitating tumor progression by modulating multiple pathways in transformed cells, including genomic stability, angiogenesis and invasion. PEG-3 originates from mutation in the growth arrest and DNA damage inducible gene GADD34. A one base deletion in rat GADD34 results in a frame-shift and premature appearance of a stop-codon resulting in a C-terminally truncated molecule that is PEG-3. We now document that mutation in the GADD34 gene is a frequent event during transformation and/or immortalization of rodent cells. Sequencing of the GADD34 gene in a number of independent rat tumor cell lines revealed that in a majority of these the GADD34 gene is mutated to either PEG-3 or a PEG-3-like gene with similar C-terminal truncations. An important function of GADD34 is to inhibit cell growth, predominantly by apoptosis, and we demonstrate that PEG-3 or C-terminal truncations of human GADD34 resembling PEG-3 prevent growth inhibition by both human and rat GADD34. Phosphorylation of p53 by GADD34 is one mechanism by which it inhibits growth and PEG-3 could prevent GADD34-induced p53 phosphorylation. In contrast, PEG-3 was unable to block other GADD34-induced changes, including eIF2adephosphorylation, indicating that its effects on GADD34 may be related more to its effect on cell growth rather than a global inhibitor of all GADD34 functions. We hypothesize that mutational generation of PEG-3 or a similar molecule is a critical event during rodent carcinogenesis. The inherent property of PEG-3 to function as a dominant negative of the growth inhibitory property of GADD34 might rescue cells from DNA damage-induced apoptosis leading to growth independence and tumorigenesis.Oncogene (2005) 24, 2247-2255. doi:10.1038/sj.onc.1208420 Published online 17 January 2005 [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is a frequent and aggressive glial tumor, containing a small population of therapy-resistant cells, glioma stem cells (GSCs). Current dogma suggests that tumors regrow from GSCs, and these cells contribute to therapy resistance, poor prognosis, and recurrence; highlighting the importance of GSCs in glioma pathophysiology and therapeutic targeting. Macroautophagy/autophagy-based cellular homeostasis can be changed from pro-survival to pro-cell death by modulating SDCBP/MDA-9/Syntenin (syndecan binding protein)-mediated signaling. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of SDCBP/MDA-9/Syntenin. Conversely, SDCBP/MDA-9/Syntenin silencing induces autophagic death in GSCs, indicating that SDCBP/MDA-9/Syntenin regulates protective autophagy in GSCs under anoikis conditions. This process is mediated through phosphorylation of the anti-apoptotic protein BCL2 accompanied with suppression of high levels of autophagic proteins (ATG5, LAMP1, LC3B) through EGFR signaling. SDCBP/MDA-9/Syntenin-mediated regulation of BCL2 and EGFR phosphorylation is achieved through PTK2/FAK and PRKC/PKC signaling. When SDCBP/MDA-9/Syntenin is absent, this protective mechanism is deregulated, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. Our recent paper reveals a novel functional link between SDCBP/MDA-9/Syntenin expression and protective autophagy in GSCs. These new insights into SDCBP/MDA-9/Syntenin-mediated regulation and maintenance of GSCs present leads for developing innovative combinatorial cancer therapies. [ABSTRACT FROM AUTHOR]

Polyinosine--polycytidylic acid [pIC] is a synthetic dsRNA that acts as an immune agonist of TLR3 and RLR to activate dendritic and natural killer cells that can kill tumor cells. pIC can also trigger apoptosis in pancreatic ductal adenocarcinoma cells (PDAC) but its mechanism of action is obscure. In this study, we investigated the potential therapeutic activity of a formulation of pIC with polyethylenimine ([pIC]PEI) in PDAC and investigated its mechanism of action. [pIC]PEI stimulated apoptosis in PDAC cells without affecting normal pancreatic epithelial cells. Mechanistically, [pIC]PEI repressed XIAP and survivin expression and activated an immune response by inducing MDA-5, RIG-I, and NOXA. Phosphorylation of AKT was inhibited by [pIC]PEI in PDAC, and this event was critical for stimulating apoptosis through XIAP and survivin degradation. In vivo administration of [pIC]PEI inhibited tumor growth via AKT-mediated XIAP degradation in both subcutaneous and quasi-orthotopic models of PDAC. Taken together, these results offer a preclinical proof-of-concept for the evaluation of [pIC]PEI as an immunochemotherapy to treat pancreatic cancer. [ABSTRACT FROM AUTHOR]

Abstract: The cytokine melanoma differentiation associated gene 7 (mda-7) was identified by subtractive hybridization as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, was classified as a member of the interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and rapid cell death. In addition, MDA-7/IL-24 has been noted to radiosensitize tumor cells which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress and suppress protein translation. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, strongly arguing that MDA-7/IL-24 could have significant therapeutic value. This review describes what is presently known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications. [ABSTRACT FROM AUTHOR]

SARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN) was identified as a novel tumor suppressor by applying subtraction hybridization to terminally differentiating human melanoma cells. The anti-tumor activity of SARI and the correlation between expression and cancer aggression and metastasis has been examined in multiple cancers, but its potential role in oral squamous cell carcinomas (OSCC) has not been explored. SARI expression was monitored in tumor tissues of OSCC patients by performing immunohistochemistry. Ectopic expression of SARI was achieved using a replication defective adenovirus expressing SARI (Ad. SARI). A nude mouse xenograft model was used to evaluate the in vivo efficacy of SARI. Endoplasmic reticulum (ER) stress was monitored in SARI infected OSCC cells by confocal microscopy. In this study, we demonstrate that SARI expression is significantly lower in OSCC tumor tissue as compared to normal adjacent tissue. Ectopic expression of SARI induces cancer-specific cell death in human OSCC cell lines and in a paclitaxel plus cisplatin non-responder OSCC patient-derived (PDC1) cell line. Mechanistically, SARI inhibits zinc finger protein GLI1 expression through induction of endoplasmic reticulum (ER) stress. Using a nude mouse xenograft model, we show that intratumoral injections of Ad. SARI significantly reduce PDC1 tumor burden, whereas treatment with an ER stress inhibitor efficiently rescues tumors from growth inhibition. Overall, our data provides a link between induction of ER stress and inhibition of the GLI1/Hedgehog signaling pathway and the tumor suppressive activity of SARI in the context of OSCC. [ABSTRACT FROM AUTHOR]

Autophagy can either be cytoprotective or promote cell death in a context-dependent manner in response to stress. How autophagy leads to autophagy dependent cell death requires further clarification. In this study, we document a nonlinear roller coaster form of autophagy oscillation when cells are subjected to different stress conditions. Serum starvation induces an initial primary autophagic peak at 6 h, that helps to replenish cells with de novo fluxed nutrients, but protracted stress lead to a secondary autophagic peak around 48 h. Time kinetic studies indicate that the primary autophagic peak is reversible, whereas the secondary autophagic peak is irreversible and leads to cell death. Key players involved in different stages of autophagy including initiation, elongation and degradation during this oscillatory sequence were identified. A similar molecular pattern was intensified under apoptosis-deficient conditions. mTOR was the central molecule regulating this autophagic activity, and upon knockdown a steady increase of autophagy without any non-linear fluctuation was evident. An unbiased proteome screening approach was employed to identify the autophagy molecules potentially regulating these autophagic peaks. Our proteomics analysis has identified Annexin A2 as a stress-induced protein to implicate in autophagy fluctuation and its deficiency reduced autophagy. Moreover, we report that mTOR in its phosphorylated condition interacts with Annexin A2 to induce autophagy fluctuation by altering its cellular localization. The work highlights the molecular mechanism of a mTOR-dependent roller coaster fluctuation of autophagy and autophagy dependent cell death during prolong stress. • Stress creates a roller coaster form of biphasic autophagic fluctuation. • Stress beyond point of no return leads to autophagy dependent cell death. • Autophagic point of no return is modulated by mTOR. • mTOR-Annexin A2 plays significant role in autophagic point of no return. [ABSTRACT FROM AUTHOR]