Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis and modulation of antitumor immune responses. In our study, we elucidated the role of MDA-7/IL-24 in inhibiting growth of breast cancer-initiating/stem cells. Ad.mda-7 infection decreased proliferation of breast cancer-initiating/stem cells without affecting normal breast stem cells. Ad.mda-7 induced apoptosis and endoplasmic reticulum stress in breast cancer-initiating/stem cells similar to unsorted breast cancer cells and inhibited the self-renewal property of breast cancer-initiating/stem cells by suppressing Wnt/β-catenin signaling. Prevention of inhibition of Wnt signaling by LiCl increased cell survival upon Ad.mda-7 treatment, suggesting that Wnt signaling inhibition might play a key role in MDA-7/IL-24-mediated death of breast cancer-initiating/stem cells. In a nude mouse subcutaneous xenograft model, Ad.mda-7 injection profoundly inhibited growth of tumors generated from breast cancer-initiating/stem cells and also exerted a potent “bystander” activity inhibiting growth of distant uninjected tumors. Further studies revealed that tumor growth inhibition by Ad.mda-7 was associated with a decrease in proliferation and angiogenesis, two intrinsic features of MDA-7/IL-24, and a reduction in vivo in the percentage of breast cancer-initiating/stem cells. Our findings demonstrate that MDA-7/IL-24 is not only nontoxic to normal cells and normal stem cells but also can kill both unsorted cancer cells and enriched populations of cancer-initiating/stem cells, providing further documentation that MDA-7/IL-24 might be a safe and effective way to eradicate cancers and also potentially establish disease-free survival.
MDA-7/IL-24; apoptosis; Wnt signaling; cancer-initiating/stem cells; breast cancer
Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting.
Subtraction-hybridization combined with induction of cancer cell terminal differentiation in human melanoma cells identified melanoma differentiation associated gene-7 (mda-7/IL-24) and SARI (Suppressor of AP-1, induced by IFN) that display potent antitumor activity. These genes are not constitutively expressed in cancer cells and forced expression of mda-7/IL-24 (Ad.mda-7) or SARI(Ad.SARI) promotes cancer-specific cell death. Ectopic expression of mda-7/IL-24 induces SARI mRNA and protein in a panel of different cancer cells leading to cell death, without harming corresponding normal cells. Simultaneous inhibition of K-ras downstream extracellular regulated kinase 1/2 (ERK1/2) signaling in pancreatic cancer cells reverses the translational block of MDA-7/IL-24 and induces SARI expression and cell death. Using SARI-antisense-based approaches we demonstrate that SARI expression is necessary for mda-7/IL-24 antitumor effects. Secreted MDA-7/IL-24 protein induces antitumor ‘bystander’ effects by promoting its own expression. Recombinant MDA-7/IL-24 (His-MDA-7) induces SARI expression, supporting the involvement of SARI in the MDA-7/IL-24-driven autocrine loop culminating in antitumor effects. Moreover, His-MDA-7 after binding to its cognate receptors (IL-20R1/IL-20R2 or IL-22R/IL-20R2) induces intracellular signaling by phosphorylation of p38 MAPK leading to transcription of a family of growth arrest and DNA damage inducible (GADD) genes, culminating in apoptosis. Inhibition of p38 MAPK fails to induce SARI following Ad.mda-7 infection. These findings reveal the significance of the mda-7/IL-24-SARI axis in cancer-specific killing, and provide a potential strategy for treating both local and metastatic disease.
SARI; MDA-7/IL-24; apoptosis; IL-20/IL-22 receptors
The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene–9 (MDA-9/syntenin) is a highly conserved PDZ domain–containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis.
We assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo.
MDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo.
Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.
MDA-9/syntenin; GBM; glioma; invasion; intracranial injection
MDA-9 (melanoma differentiation associated gene-9)/Syntenin is a PDZ domain-containing adaptor protein involved in multiple diverse cellular processes including organization of protein complexes in the plasma membrane, intracellular trafficking and cell surface targeting, synaptic transmission, and cancer metastasis. In the present study, we analyzed the expression pattern of MDA-9/syntenin during mouse development. MDA-9/syntenin was robustly expressed with tight regulation of its temporal and spatial expression during fetal development in the developing skin, spinal cord, heart, lung and liver, which are regulated by multiple signaling pathways in the process of organogenesis. Recent studies also indicate that MDA-9/syntenin is involved in the signaling pathways crucial during development such as Wnt, Notch and FGF. Taken together, these results suggest that MDA-9/syntenin may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation through modulating multiple signaling pathways as a crucial adaptor protein. Additionally, temporal regulation of MDA-9/syntenin expression may be required during specific stages and in specific tissues during development.
MDA-9/syntenin; development; mouse embryo; adaptor protein
Programmed cell death is well-orchestrated process regulated by multiple pro-apoptotic and anti-apoptotic genes, particularly those of the Bcl-2 gene family. These genes are well documented in cancer with aberrant expression being strongly associated with resistance to chemotherapy and radiation.
This review focuses on the resistance induced by the Bcl-2 family of anti-apoptotic proteins and current therapeutic interventions currently in preclinical or clinical trials that target this pathway. Major resistance mechanisms that are regulated by Bcl-2 family proteins and potential strategies to circumvent resistance are also examined. Although antisense and gene therapy strategies are used to nullify Bcl-2 family proteins, recent approaches use small molecule inhibitors and peptides. Structural similarity of the Bcl-2 family of proteins greatly favors development of inhibitors that target the BH3 domain, called BH3 mimetics.
Strategies to specifically identify and inhibit critical determinants that promote therapy-resistance and tumor progression represent viable approaches for developing effective cancer therapies. From a clinical perspective, pretreatment with novel, potent Bcl-2 inhibitors either alone or in combination with conventional therapies hold significant promise for providing beneficial clinical outcomes. Identifying small molecule inhibitors with broader and higher affinities for inhibiting all of the Bcl-2 pro-survival proteins will facilitate development of superior cancer therapies.
BH3 domain; apoptosis; Mcl-1; radiation resistance; chemotherapy resistance
Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression.
Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knock down was examined in multiple cells lines and key findings were validated in primary tumors.
Significantly higher (p= 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28/44) of primary tumors and an association was evident with stage (p=0.01), grade (p=0.03) and invasion status (p=0.02). MDA-9/Syntenin overexpression in non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and co-localization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin: AKT expressions with stage (p=0.04, EGFR), (p=0.01, AKT). A correlation between MDA-9/Syntenin: β-catenin co-expression with stage (p=0.03) and invasion (p=0.04) was also evident.
Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring and therapeutic strategies for managing UCC.
Urothelial cancer; MDA-9/Syntenin; invasion; EGFR signaling
Efficient cross-presentation of protein antigens to cytotoxic T lymphocytes (CTLs) by dendritic cells (DCs) is essential for the success of prophylactic and therapeutic vaccines. Here, we report a previously underappreciated pathway involving antigen entry into the endoplasmic reticulum (ER) critically needed for T-cell cross-priming induced by a DC-targeted vaccine. Directing the clinically relevant, melanoma antigen Gp100 to mouse-derived DCs by molecular adjuvant and chaperone Grp170 substantially facilitates antigen access to the ER. Grp170 also strengthens the interaction of internalized protein antigen with molecular components involved in ER-associated protein dislocation and/or degradation, which culminates in cytosolic translocation for proteasome-dependent degradation and processing. Targeted disruption of protein retrotranslocation causes exclusive ER retention of tumor antigen in mouse bone marrow-derived DCs and splenic CD8+ DCs. This results in the blockade of antigen ubiquitination and processing, which abrogates the priming of antigen-specific CD8+ T cells in vitro and in vivo. Therefore, the improved ER entry of tumor antigen serves as a molecular basis for the superior cross-presenting capacity of Grp170-based vaccine platform. The ER access and retrotranslocation represents a distinct pathway that operates within DCs for cross-presentation, and is required for the activation of antigen-specific CTLs by certain vaccines. These results also reinforce the importance of the ER-associated protein quality-control machinery and the mode of the antigen delivery in regulating DC-elicited immune outcomes.
dendritic cell; cross-presentation; endoplasmic reticulum; vaccine; melanoma antigen; molecular chaperone
The staphylococcal nuclease and tudor domain containing 1 (SND1) is a multifunctional protein overexpressed in breast, prostate, colorectal and hepatocellular carcinomas and malignant glioma. Molecular studies have revealed the multifaceted activities of SND1 involved in regulating gene expression at transcriptional as well as post-transcriptional levels. Early studies identified SND1 as a transcriptional co-activator. SND1 is also a component of RNA-induced silencing complex (RISC) thus mediating RNAi function, a regulator of mRNA splicing, editing and stability, and plays a role in maintenance of cell viability. Such diverse actions allow the SND1 to modulate a complex array of molecular networks, thereby promoting carcinogenesis. Here, we describe the crucial role of SND1 in cancer development and progression, and highlight SND1 as a potential target for therapeutic intervention.
staphylococcal nuclease and tudor domain containing 1; astrocyte elevated gene-1; cancer; metastasis
We describe a new imaging method for detecting prostate cancer, whether localized or disseminated and metastatic to soft tissues and bone. The method relies on the use of imaging reporter genes under the control of the promoter of AEG-1 (MTDH), which is selectively active only in malignant cells. Through systemic, nanoparticle-based delivery of the imaging construct, lesions can be identified through bioluminescence imaging and single photon emission-computed tomography in the PC3-ML murine model of prostate cancer at high sensitivity. This approach is applicable for the detection of prostate cancer metastases, including bone lesions for which there is no current reliable agent for non-invasive clinical imaging. Further, the approach compares favorably to accepted and emerging clinical standards, including positron emission tomography with [18F]fluorodeoxyglucose and [18F]sodium fluoride. Our results offer a preclinical proof of concept that rationalizes clinical evaluation in patients with advanced prostate cancer.
molecular-genetic imaging; bioluminescence; SPECT; metastasis; nanoparticle; PC3
Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored an hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested crosstalk between these two proteins. Further, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Lastly, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.
RKIP; MDA-9/syntenin, melanoma; c-Src; FAK
The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL-2 family inhibitor) to enhance [sorafenib + HDAC inhibitor] toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of [sorafenib + HDAC inhibitor] exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K and mTOR activity and enhanced cleavage of pro-caspase 3 and reduced expression of MCL-1 and BCL-XL. Over-expression of BCL-XL or MCL-1 or expression of dominant negative pro-caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTORand to a lesser extent MEK1EE also protected cells that correlated with maintained c-FLIP-s expression, reduced BIM expression and increased BAD phosphorylation. In vivo [sorafenib + HDAC inhibitor] toxicity against tumors was increased in a greater than additive fashion by TRAIL.Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting [sorafenib + HDAC inhibitor] lethality.
sorafenib; histone deacetylase inhibitor; kinase; apoptosis; CD95; TRAIL; MCL-1
Melanoma differentiation associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacological approaches were employed to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, CAM assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several pro-angiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the ECM activating Src and FAK resulting in activation by phosphorylation of Akt, which induces HIF-1α. The HIF-1α activates transcription of Insulin Growth Factor Binding Protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell non-autonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (non-autonomous).
mda-9/syntenin; melanoma; angiogenesis; IGFBP-2; HuVECs; CAM assay
Little is known about the molecular pathways regulating poor differentiation and invasion of head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to determine the role of MDA-9/Syntenin, a metastasis associated molecule in HNSCC tumorigenesis. Elevated MDA-9/Syntenin expression was evident in 67% (54/81) primary HNSCC tumors (p=0.001-0.002) and 69% (9/13) pre-neoplastic tissues (p=0.02-0.03). MDA-9/Syntenin overexpression was associated with the stage (p=0.001), grade (p=0.001) and lymph node metastasis (p=0.0001). Silencing of MDA-9/Syntenin in 3 poorly differentiated HNSCC cell lines induced squamous epithelial cell differentiation, disrupted angiogenesis and reduced tumor growth in vitro and in vivo. We confirmed SPRR1B and VEGFR1 as the key molecular targets of MDA-9/Syntenin on influencing HNSCC differentiation and angiogenesis respectively. MDA-9/Syntenin disrupted SPRR1B expression interacting through its PDZ1 domain and altered VEGFR1 expression in vitro and in vivo. VEGFR1 co-localized with MDA-9/Syntenin in HNSCC cell lines and primary tumor. Downregulation of growth regulatory molecules CyclinD1, CDK4, STAT3, PI3K and CTNNB1 was also evident in the MDA-9/Syntenin depleted cells, which was reversed following over-expression of MDA-9/Syntenin in immortalized oral epithelial cells. Our results suggest that early induction of MDA-9/Syntenin expression influences HNSCC progression and should be further evaluated for potential biomarker development.
Head and neck suqamous cell carcinoma; MDA-9/Syntenin; differentiation; SPRR1B; VEGFR1
Aggressive tumor growth, diffuse tissue invasion and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. AEG-1 is an oncogene overexpressed in multiple types of human cancers including >90% of brain tumors. AEG-1 also promotes gliomagenesis particularly in the context of tumor growth and invasion, two primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and glioma patient samples indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain and loss of function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in glioma patient samples demonstrating that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.
AEG-1; glioma; EAAT2; glutamate; glioma-induced neurodegeneration
RIG-I-like receptors (RLRs) MDA5 and RIG-I are key players in the innate antiviral response. Upon recognition of viral RNA, they interact with MAVS, eventually inducing type I interferon production. The interferon induction pathway is commonly targeted by viruses. How enteroviruses suppress interferon production is incompletely understood. MDA5 has been suggested to undergo caspase- and proteasome-mediated degradation during poliovirus infection. Additionally, MAVS is reported to be cleaved during infection with coxsackievirus B3 (CVB3) by the CVB3 proteinase 3Cpro, whereas MAVS cleavage by enterovirus 71 has been attributed to 2Apro. As yet, a detailed examination of the RLR pathway as a whole during any enterovirus infection is lacking. We performed a comprehensive analysis of crucial factors of the RLR pathway, including MDA5, RIG-I, LGP2, MAVS, TBK1, and IRF3, during infection of CVB3, a human enterovirus B (HEV-B) species member. We show that CVB3 inhibits the RLR pathway upstream of TBK1 activation, as demonstrated by limited phosphorylation of TBK1 and a lack of IRF3 phosphorylation. Furthermore, we show that MDA5, MAVS, and RIG-I all undergo proteolytic degradation in CVB3-infected cells through a caspase- and proteasome-independent manner. We convincingly show that MDA5 and MAVS cleavages are both mediated by CVB3 2Apro, while RIG-I is cleaved by 3Cpro. Moreover, we show that proteinases 2Apro and 3Cpro of poliovirus (HEV-C) and enterovirus 71 (HEV-A) exert the same functions. This study identifies a critical role of 2Apro by cleaving MDA5 and MAVS and shows that enteroviruses use a common strategy to counteract the interferon response in infected cells.
IMPORTANCE Human enteroviruses (HEVs) are important pathogens that cause a variety of diseases in humans, including poliomyelitis, hand, foot, and mouth disease, viral meningitis, cardiomyopathy, and more. Like many other viruses, enteroviruses target the host immune pathways to gain replication advantage. The MDA5/MAVS pathway is responsible for recognizing enterovirus infections in the host cell and leads to expression of type I interferons (IFN-I), crucial antiviral signaling molecules. Here we show that three species of HEVs all employ the viral proteinase 2A (2Apro) to proteolytically target MDA5 and MAVS, leading to an efficient blockade upstream of IFN-I transcription. These observations suggest that MDA5/MAVS antagonization is an evolutionarily conserved and beneficial mechanism of enteroviruses. Understanding the molecular mechanisms of enterovirus immune evasion strategies will help to develop countermeasures to control infections with these viruses in the future.
The incidence of melanoma continues to rise and prognosis in patients with metastatic melanoma remains poor. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) serves as one of the primary immune checkpoints and downregulates T cell activation pathways. Enhancing T cell activation by antibody blockade of the CTLA-4 provides a novel approach to overcome tumor-induced immune tolerance. Recently, anti-CTLA-4 therapy demonstrated significant clinical benefit in patients with metastatic melanoma, which led to the approval of ipilimumab by the Food and Drug Administration in early 2011.
The fundamental concepts underlying CTLA-4 blockade-potentiated immune activation, the scientific rationale for and the preclinical evidence supporting CTLA-4-targeted cancer immunotherapy are presented. We also provide an update on clinical trials with anti-CTLA-4 inhibitors and discuss the associated autoimmune toxicity.
Given that overall survival is the only validated endpoint for the anti-CTLA-4 therapy, the clinical implications of the antigen or tumor-specific immunity in patients remain to be clarified. Additional research is necessary to elucidate the prognostic significance of immune-related side effects and significantly optimize the treatment regimens. An improved understanding of the mechanisms of action of CTLA-4 antibodies may also culminate in wide-ranging clinical applications of this novel therapy for other tumor types.
cytotoxic T-lymphocyte-associated antigen; CTL-A blockade; T cell activation; tumor immunity; overall survival
Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.
Viral gene therapy; Mcl-1 inhibition; apoptosis induction; anti-tumor activity
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a unique member of the IL-10 gene family, displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti-tumor immune responses. Here we identify clusterin (CLU) as a MDA-7/IL-24 interacting protein in DU-145 cells and investigate the role of MDA-7/IL-24 in regulating CLU expression and mediating the antitumor properties of mda-7/IL-24 in prostate cancer. Ad.mda-7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad.mda-7 infection sCLU expression increased and CLU interacted with MDA-7/IL-24 producing a cytoprotective effect. Infection of stable clones of DU-145 prostate cancer cells expressing sCLU with Ad.mda-7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda-7/IL-24, sCLU-DU-145 cells displayed G2/M phase arrest followed by apoptosis. Similarly, Ad.mda-7 infection decreased cell migration by altering cytoskeleton in sCLU-DU-145 cells. Ad.mda-7-treated sCLU-DU-145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA-7/IL-24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA-7/IL-24 that may be exploited for developing enhanced therapies for prostate cancer.
MDA-7/IL-24; soluble clusterin; nuclear clusterin; G2/M arrest; apoptosis
Despite significant improvements in therapeutic protocols, Head and Neck Squamous Cell Carcinoma (HNSCC) remains a major health problem worldwide. The 5-year post therapeutic survival rate is among the lowest of the major cancers with loco-regional relapse being the main cause of death. Moreover, in most instances, the quality of life of the afflicted patient is severely compromised. The poor prognosis for HNSCC is primarily due to disease detection at advanced stages. Accordingly, development of early detection and preventive strategies are essential. Recent advances in our understanding of the molecular biology and etiology of HNSCC should facilitate development of improved intervention and therapeutic approaches. The present review discusses the potential role of such factors for developing preventive and early diagnostic strategies for HNSCC management.
Converting the immunosuppressive tumor environment into one that is favorable to induction of antitumor immunity is indispensable for effective cancer immunotherapy. Here we strategically incorporate a pathogen (i.e., Flagellin)-derived, NF-κB-stimulating `danger' signal into the large stress protein or chaperone Grp170 (HYOU1/ORP150) that was previously shown to facilitate antigen cross-presentation. This engineered chimeric molecule (i.e., Flagrp170) is capable of transporting tumor antigens and concurrently inducing functional activation of dendritic cells. Intratumoral administration of adenoviruses expressing Flagrp170 induces a superior antitumor response against B16 melanoma and its distant lung metastasis compared to unmodified Grp170 and Flagellin. The enhanced tumor destruction is accompanied with significantly increased tumor infiltration by CD8+ cells as well as elevation of IFN-γ and IL-12 levels in the tumor sites. In situ Ad.Flagrp170 therapy provokes systemic activation of CTLs that recognize several antigens naturally expressing in melanoma (e.g., gp100/PMEL and TRP2/DCT). The mechanistic studies using CD11c-DTR transgenic mice and Batf3-deficient mice reveal that CD8α+ DCs are required for the improved T cell cross-priming. Antibody neutralization assays show that IL-12 and IFN-γ are essential for the Flagrp170-elicited antitumor response, which also involves CD8+ T cells and NK cells. The therapeutic efficacy of Flagrp170 and its immune stimulating activity are also confirmed in mouse prostate cancer and colon carcinoma. Together, targeting the tumor microenvironment with this chimeric chaperone is highly effective in mobilizing or restoring antitumor immunity, supporting the potential therapeutic use of this novel immune modulator in the treatment of metastatic diseases.
molecular chaperone; Grp170; Flagellin; immune modulator; antitumor immunity
•SND1 augments AT1R receptor level by posttranscriptional regulation.•SND1 activates TGFβ signaling which promotes the epithelial–mesenchymal transition.•Migration and invasion by human hepatocellular carcinoma (HCC) cells are augmented by SND1.•A correlation is observed between SND1 and AT1R expression in HCC patients.
Staphylococcal nuclease domain containing-1 (SND1) is overexpressed in human hepatocellular carcinoma (HCC) patients and promotes tumorigenesis by human HCC cells. We now document that SND1 increases angiotensin II type 1 receptor (AT1R) levels by increasing AT1R mRNA stability. This results in activation of ERK, Smad2 and subsequently the TGFβ signaling pathway, promoting epithelial–mesenchymal transition (EMT) and migration and invasion by human HCC cells. A positive correlation was observed between SND1 and AT1R expression levels in human HCC patients. Small molecule inhibitors of SND1, alone or in combination with AT1R blockers, might be an effective therapeutic strategy for late-stage aggressive HCC.
ACE, angiotensin-I converting enzyme; ACE-I, ACE inhibitors; AT1R, angiotensin II type 1 receptor; EMT, epithelial–mesenchymal transition; FDR, false discovery rate; HCC, human hepatocellular carcinoma; LP, losartan potassium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NASH, non-alcoholic steatohepatitis; PAI-1, plasminogen activator inhibitor-1; RISC, RNA-induced silencing complex; SND1, Staphylococcal nuclease domain containing-1; SND1; AT1R; TGFβ; PAI-1; Invasion
Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.
AEG-1; development; mouse embryo; cell proliferation; cancer
Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.
A potentially less toxic approach for cancer therapy comprises induction of tumor cells to lose growth potential irreversibly and terminally differentiate. Combining this scheme termed ‘differentiation therapy of cancer’ with subtraction hybridization to human melanoma cells resulted in the cloning of melanoma differentiation associated (mda) genes displaying elevated expression as a consequence of induction of terminal differentiation. One originally novel gene, mda-7, was found to display elevated expression in normal melanocytes and nevi with progressive loss of expression as a consequence of melanoma development and progression to metastasis. Based on structure, biochemical properties and chromosomal location, mda-7 has now been reclassified as interleukin (IL)-24 a member of the expanding IL-10 family of cytokines. In vitro cell culture and in vivo animal studies indicate that mda-7/IL-24 selectively induces programmed cell death (apoptosis) in multiple human cancers (including melanomas), without harming normal cells, and promotes profound anti-tumor activity in nude mice containing human tumor xenografts. Based on these remarkable properties, a Phase I Clinical trial was conducted to test the safety of administration of mda-7/IL-24 by a replication incompetent adenovirus (Ad.mda-7; INGN 241) in patients with advanced solid cancers including melanoma. mda-7/IL-24 was found to be safe and to promote significant clinical activity, particularly in the context of patients with metastatic melanoma. These results provide an impetus for further clinical studies, and document a central paradigm of cancer therapy, namely translation of basic science from the “bench to the bedside.”
mda-7/IL-24; apoptosis; metastatic melanoma; Phase I Clinical Trial