Melanie Mediavilla-Varela

Tumor-associated Antigen Arrays for the Serological Diagnosis of Cancer

The recognition that human tumors stimulate the production of autoantibodies against autologous cellular proteins called tumor-associated antigens (TAAs) has opened the door to the possibility that autoantibodies could be exploited as serological tools for the early diagnosis and management of cancer. Cancer-associated autoantibodies are often driven by intracellular proteins that are mutated, modified, or aberrantly expressed in tumor cells and hence are regarded as immunological reporters that could help uncover molecular events underlying tumorigenesis. Emerging evidence suggests that each type of cancer might trigger unique autoantibody signatures that reflect the nature of the malignant process in the affected organ. The advent of novel genomic, proteomic, and high throughput approaches has accelerated interest in the serum autoantibody repertoire in human cancers for the discovery of candidate TAAs. The use of individual anti-TAA autoantibodies as diagnostic or prognostic tools has been tempered by their low frequency and heterogeneity in most human cancers. However, TAA arrays comprising several antigens significantly increase this frequency and hold great promise for the early detection of cancer, monitoring cancer progression, guiding individualized therapeutic interventions, and identification of novel therapeutic targets. Our recent studies suggest that the implementation of TAA arrays in screening programs for the diagnosis of prostate cancer and other cancers should be preceded by the optimization of their sensitivity and specificity through the careful selection of the most favorable combinations of TAAs.

Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

BackgroundHormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality.ResultsWe investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity.ConclusionThese results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC.

Antagonism of adenosine A2A receptor expressed by lung adenocarcinoma tumor cells and cancer associated fibroblasts inhibits their growth

Recently it has become clear that the cost associated with the Warburg effect, which is inefficient production of ATP, is offset by selective advantages that are produced by resultant intracellular metabolic alterations. In fact tumors may be addicted to the Warburg effect. In addition these alterations result in changes in the extracellular tumor microenvironment that can also produce selective advantages for tumor cell growth and survival. One such extracellular alteration is increased adenosine concentrations that have been shown to impair T cell mediated rejection and support angiogenesis. The expression of the A2A receptor in non-small cell cancer (NSCLC) tissues, cell lines and cancer associated fibroblasts (CAF) was determined by performing immunohistrochemistry and immunoblot analysis. The efficacy of the A2A receptor antagonists in vivo was evaluated in a PC9 xenograft model. To determine the mode of cell death induced by A2A receptor antagonists flow cytometry, immunoblot, and cytotoxic analysis were performed. We found that a significant number of lung adenocarcinomas express adenosine A2A receptors. Antagonism of these receptors impaired CAF and tumor cell growth in vitro and inhibited human tumor xenograft growth in mice. These observations add to the rationale for testing adenosine A2A receptor antagonists as anticancer therapeutics. Not only could there be prevention of negative signaling in T cells within the tumor microenvironment and inhibition of angiogenesis, but also an inhibitory effect on tumor-promoting, immunosuppressive CAFs and a direct inhibitory effect on the tumor cells themselves.

Alternative Splicing and Caspase-Mediated Cleavage Generate Antagonistic Variants of the Stress Oncoprotein LEDGF/p75

There is increasing evidence that an augmented state of cellular oxidative stress modulates the expression of stress genes implicated in diseases associated with health disparities such as certain cancers and diabetes. Lens epithelium–derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, is emerging as a survival oncoprotein that promotes resistance to oxidative stress–induced cell death and chemotherapy. We previously showed that LEDGF/p75 is targeted by autoantibodies in prostate cancer patients and is overexpressed in prostate tumors, and that its stress survival activity is abrogated during apoptosis. LEDGF/p75 has a COOH-terminally truncated splice variant, p52, whose role in stress survival and apoptosis has not been thoroughly investigated. We observed unbalanced expression of these proteins in a panel of tumor cell lines, with LEDGF/p75 generally expressed at higher levels. During apoptosis, caspase-3 cleaved p52 to generate a p38 fragment that lacked the NH2-terminal PWWP domain and failed to transactivate the Hsp27 promoter in reporter assays. However, p38 retained chromatin association properties and repressed the transactivation potential of LEDGF/p75. Overexpression of p52 or its variants with truncated PWWP domains in several tumor cell lines induced apoptosis, an activity that was linked to the presence of an intron-derived COOH-terminal sequence. These results implicate the PWWP domain of p52 in transcription function but not in chromatin association and proapoptotic activities. Consistent with their unbalanced expression in tumor cells, LEDGF/p75 and p52 seem to play antagonistic roles in the cellular stress response and could serve as targets for novel antitumor therapies. (Mol Cancer Res 2008;6(8):1293–307)

LEDGF/p75 Overexpression Attenuates Oxidative Stress-Induced Necrosis and Upregulates the Oxidoreductase ERP57/PDIA3/GRP58 in Prostate Cancer

Prostate cancer (PCa) mortality is driven by highly aggressive tumors characterized by metastasis and resistance to therapy, and this aggressiveness is mediated by numerous factors, including activation of stress survival pathways in the pro-inflammatory tumor microenvironment. LEDGF/p75, also known as the DFS70 autoantigen, is a stress transcription co-activator implicated in cancer, HIV-AIDS, and autoimmunity. This protein is targeted by autoantibodies in certain subsets of patients with PCa and inflammatory conditions, as well as in some apparently healthy individuals. LEDGF/p75 is overexpressed in PCa and other cancers, and promotes resistance to chemotherapy-induced cell death via the transactivation of survival proteins. We report in this study that overexpression of LEDGF/p75 in PCa cells attenuates oxidative stress-induced necrosis but not staurosporine-induced apoptosis. This finding was consistent with the observation that while LEDGF/p75 was robustly cleaved in apoptotic cells into a p65 fragment that lacks stress survival activity, it remained relatively intact in necrotic cells. Overexpression of LEDGF/p75 in PCa cells led to the upregulation of transcript and protein levels of the thiol-oxidoreductase ERp57 (also known as GRP58 and PDIA3), whereas its depletion led to ERp57 transcript downregulation. Chromatin immunoprecipitation and transcription reporter assays showed LEDGF/p75 binding to and transactivating the ERp57 promoter, respectively. Immunohistochemical analysis revealed significantly elevated co-expression of these two proteins in clinical prostate tumor tissues. Our results suggest that LEDGF/p75 is not an inhibitor of apoptosis but rather an antagonist of oxidative stress-induced necrosis, and that its overexpression in PCa leads to ERp57 upregulation. These findings are of significance in clarifying the role of the LEDGF/p75 stress survival pathway in PCa.

Transforming Growth Factor β Signaling Overcomes Dasatinib Resistance in Lung Cancer

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFβ type I receptor (TβR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFβ on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFβ and dasatinib, apoptosis was induced. Combined TGFβ-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFβ intracellular mediators. Interestingly, combined TGFβ and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFβ-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

A GM-CSF and CD40L bystander vaccine is effective in a murine breast cancer model.

Background There is increasing interest in using cancer vaccines to treat breast cancer patients in the adjuvant setting to prevent recurrence in high risk situations or in combination with other immunomodulators in the advanced setting. Current peptide vaccines are limited by immunologic compatibility issues, and engineered autologous cellular vaccines are difficult to produce on a large scale. Using standardized bystander cell lines modified to secrete immune stimulating adjuvant substances can greatly enhance the ability to produce immunogenic cellular vaccines using unmodified autologous cells or allogeneic medical grade tumor cell lines as targets. We investigated the efficacy of a cellular vaccine using B78H1 bystander cell lines engineered to secrete granulocyte macrophage-colony stimulating factor and CD40 ligand (BCG) in a murine model of breast cancer. Methods Five-week-old female BALB/c mice were injected orthotopically in the mammary fat pad with 4T1 tumor cells. Treatment consisted of irradiated 4T1 ± BCG cells given subcutaneously every 4 days and was repeated three times per mouse when tumors became palpable. Tumors were measured two to three times per week for 25 days. The vaccine’s activity was confirmed in a second experiment using Lewis lung carcinoma (LLC) cells in C57BL/6 mice to exclude a model specific effect. Interferon-γ (IFN-γ) and interleukin-2 (IL-2) enzyme-linked immunospots (ELISPOTS) were performed on splenic lymphocytes incubated with 4T1 lysates along with immunohistochemistry for CD3 on tumor sections. Results Tumor growth was significantly inhibited in the 4T1-BCG and LLC-BCG treatment groups when compared to 4T1 and LLC treatment groups. There were higher levels of IL-2 and IFN-γ secreting T-cells on ELISPOT for BCG treated groups, and a trend for higher numbers of tumor infiltrating CD3+ lymphocytes. Some tumors in the 4T1-BCG demonstrated organized lymphoid structures within the tumor microenvironment as well. Conclusion The use of BCG bystander cell lines demonstrates proof of concept for anti-tumor activity and immunogenicity in an immunocompetent murine model of breast cancer. This vaccine is being evaluated in lung cancer and should be explored further in clinical trials of breast cancer patients at high risk of recurrence or in combination with other immunomodulatory agents.

Abstract 4948: Pirfenidone as a potential therapeutic treatment for CAFs and cancer cells in NSCLC.

Lung cancer is the leading cause of cancer death in the United States. Due to limited efficacy of current conventional chemotherapy, the majority of patients face poor prognosis and eventually succumb the disease. Until recently, the main focus in cancer research has been targeting the neoplastic cell. However, the importance of the tumor microenvironment, including cancer associated fibroblasts (CAFs), in cancer progression is now clear and targeting both, the neoplastic cell as well as the CAF, is being recognized as an important therapeutic approach. We have recently discovered that pirfenidone might be the drug to target these cells. Pirfenidone is a small molecule that has been shown to decrease fibrosis in a variety of fibrotic diseases. The exact mechanism responsible for its anti-fibrotic effects remains to be elucidated. Our hypothesis is that pirfenidone (by targeting the CAFs) in combination with standard chemotherapy (by targeting the cancer cells) will act synergistically and proffer a more potent therapeutic strategy for non-small cell lung cancer (NSCLC). Our studies show that in the presence of pirfenidone NSCLC associated CAF viability decreases in vitro. Furthermore, pirfenidone was able to induce apoptotic cell death as well as cell cycle arrest in NSCLC associated CAFs. In addition, we were able to observe that CAFs treated with pirfenidone decreased active TGFβ when compared to the control. Moreover, in a spheroid model, pirfenidone was able to affect the crosstalk between the cancer cells and CAFs leading to increased apoptosis and decreased viability. Currently, pirfenidone is being used in Phase II and III clinical trials for idiopathic pulmonary fibrosis. Our lab is the first one to recognize the potential of pirfenidone as a potential treatment of NSCLC. Citation Format: Melanie Mediavilla-Varela, Kimberly Luddy, David Noyes, Kingsley Boateng, Scott Antonia. Pirfenidone as a potential therapeutic treatment for CAFs and cancer cells in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2013-4948

A phase I/randomized phase II study of GM.CD40L vaccine in combination with CCL21 in patients with advanced lung adenocarcinoma

The GM.CD40L vaccine, which recruits and activates dendritic cells, migrates to lymph nodes, activating T cells and leading to systemic tumor cell killing. When combined with the CCL21 chemokine, which recruits T cells and enhances T-cell responses, additive effects have been demonstrated in non-small cell lung cancer mouse models. Here, we compared GM.CD40L versus GM.CD40L plus CCL21 (GM.CD40L.CCL21) in lung adenocarcinoma patients with ≥ 1 line of treatment. In this phase I/II randomized trial (NCT01433172), patients received intradermal vaccines every 14 days (3 doses) and then monthly (3 doses). A two-stage minimax design was used. During phase I, no dose-limiting toxicities were shown in three patients who received GM.CD40L.CCL21. During phase II, of evaluable patients, 5/33 patients (15.2%) randomized for GM.DCD40L (p = .023) and 3/32 patients (9.4%) randomized for GM.DCD40L.CCL21 (p = .20) showed 6-month progression-free survival. Median overall survival was 9.3 versus 9.5 months with GM.DCD40L versus GM.DCD40L.CCL21 (95% CI 0.70–2.25; p = .44). For GM.CD40L versus GM.CD40L.CCL21, the most common treatment-related adverse events (TRAEs) were grade 1/2 injection site reaction (51.4% versus 61.1%) and grade 1/2 fatigue (35.1% versus 47.2%). Grade 1 immune-mediated TRAEs were isolated to skin. No patients showed evidence of pseudo-progression or immune-related TRAEs of grade 1 or greater of pneumonitis, endocrinopathy, or colitis, and none discontinued treatment due to toxicity. Although we found no significant associations between vaccine immunogenicity and outcomes, in limited biopsies, one patient treated with GMCD40L.CCL21 displayed abundant tumor-infiltrating lymphocytes. This possible effectiveness warrants further investigation of GM.CD40L in combination approaches.

Abstract 1704: Combination of Pirfenidone and Cisplatin as a potential therapeutic strategy in NSCLC

Lung cancer is the leading cause of cancer death in the United States. Due to limited efficacy of current conventional chemotherapy, the majority of patients face poor prognosis and eventually succumb the disease. Therefore, the study of combination therapy using traditional anticancer drugs with novel therapeutic approaches is needed. Pirfenidone is a small molecule that is being used in Phase II and III clinical trials for idiopathic pulmonary fibrosis (IPF). Although it has already been approved for IPF in Europe, there is no known mechanism of action known for the drug. Recently we discovered that Pirfenidone has a detrimental effect on cancer associated fibroblasts (CAFs) as well as in non-small cell lung cancer (NSCLC) cells. Our hypothesis is that Pirfenidone, in combination with standard chemotherapy, will act synergistically and proffer a more potent therapeutic strategy for NSCLC. Our studies show that in a co-culture model there is a decrease in viability on CAFs and NSCLC cells when the combination of Pirfenidone and Cisplatin is used. Furthermore, Pirfenidone and Cisplatin were able to have an anti-tumor effect in a xenograft model of NSCLC and CAFs. In addition, insights in the mechanism by which Pirfenidone and Cisplatin induce synergy in NSCLC cells are being elucidated. Our studies show that the combination of drugs leads to an increase in phosphorylated ERK as well as a decrease in phosphorylated Akt. Thus, involving the PI3K and MAPK/ERK pathways in Pirfenidone/Cisplatin induced cell death. Our lab is the first to recognize the therapeutic potential of combining Pirfenidone and Cisplatin, as well as elucidating the mechanism of action for both drugs in NSCLC. Citation Format: Melanie Mediavilla-Varela, David Noyes, Kingsley Boateng, Scott Antonia. Combination of Pirfenidone and Cisplatin as a potential therapeutic strategy in NSCLC. [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 1704. doi:10.1158/1538-7445.AM2014-1704