IFNγ Modulates the Immunopeptidome of Triple Negative Breast Cancer Cells by Enhancing and Diversifying Antigen Processing and Presentation.

Peptide vaccination remains a viable approach to induce T-cell mediated killing of tumors. To identify potential T-cell targets for Triple-Negative Breast Cancer (TNBC) vaccination, we examined the effect of the pro-inflammatory cytokine interferon-γ (IFNγ) on the transcriptome, proteome, and immunopeptidome of the TNBC cell line MDA-MB-231. Using high resolution mass spectrometry, we identified a total of 84,131 peptides from 9,647 source proteins presented by human leukocyte antigen (HLA)-I and HLA-II alleles. Treatment with IFNγ resulted in a remarkable remolding of the immunopeptidome, with only a 34% overlap between untreated and treated cells across the HLA-I immunopeptidome, and expression of HLA-II only detected on treated cells. IFNγ increased the overall number, diversity, and abundance of peptides contained within the immunopeptidome, as well increasing the coverage of individual source antigens. The suite of peptides displayed under conditions of IFNγ treatment included many known tumor associated antigens, with the HLA-II repertoire sampling 17 breast cancer associated antigens absent from those sampled by HLA-I molecules. Quantitative analysis of the transcriptome (10,248 transcripts) and proteome (6,783 proteins) of these cells revealed 229 common proteins and transcripts that were differentially expressed. Most of these represented downstream targets of IFNγ signaling including components of the antigen processing machinery such as tapasin and HLA molecules. However, these changes in protein expression did not explain the dramatic modulation of the immunopeptidome following IFNγ treatment. These results demonstrate the high degree of plasticity in the immunopeptidome of TNBC cells following cytokine stimulation and provide evidence that under pro-inflammatory conditions a greater variety of potential HLA-I and HLA-II vaccine targets are unveiled to the immune system. This has important implications for the development of personalized cancer vaccination strategies.

Investigation on the Anticancer Activity of Symmetric and Unsymmetric Cyclic Sulfamides.

The sulfamide functional group has been extensively employed in organic synthesis to discover probes and drugs in various applications such as cancer, human immunodeficiency virus (HIV), virus, and diabetes. Herein, we describe the synthesis of 7-membered symmetric and unsymmetric sulfamide compounds and their biological evaluation through the National Cancer Institute (NCI) panel of 60 human tumor cell lines (NCI-60) and the mechanism of action study. The results of a study from the NCI-60 cell line exhibited that many synthesized cyclic sulfamide compounds inhibited breast cancer (MDA-MB-468). The mechanism of action study of a representative compound 18 showed the inhibition of proliferation and apoptosis in A549 lung cancer cells.

Spliced Peptides and Cytokine-Driven Changes in the Immunopeptidome of Melanoma.

Antigen recognition by CD8+ T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of noncontiguous regions of proteins to form novel peptide antigens. Here, we used high-resolution mass spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of IFNγ. In total, we identified more than 60,000 peptides from a single patient-derived cell line (LM-MEL-44) and demonstrated that IFNγ induced changes in the peptidome, with an overlap of only approximately 50% between basal and treated cells. Around 6% to 8% of the peptides were identified as cis-spliced peptides, and 2,213 peptides (1,827 linear and 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive- and IFNγ-induced peptidome. We next examined additional HLA-matched patient-derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides was conserved between individual patient tumors, drawing on data amassing to more than 100,000 peptide sequences. Several of these peptides showed in vitro immunogenicity across multiple patients with melanoma. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major class of tumor antigens.

Interleukin-6 induced overexpression of valosin-containing protein (VCP)/p97 is associated with androgen-independent prostate cancer (AIPC) progression.

Though Androgen deprivation therapy (ADT) is effective initially, numerous patients become resistant to it and develop castration resistant PCa (CRPC). Cytokines promotes ligand independent activation of AR. Interleukin-6 (IL-6) levels are elevated in CRPC patients and regulate AR activity. However, progression to CRPC is not fully understood. In this study, we analyzed differential protein expression in LNCaP cells treated with IL-6 using proteomics. Results revealed altered expression of 27 proteins and Valosin-containing protein (VCP)/p97 plays a predominant role in co-regulation of altered proteins. Interestingly, IL-6 induced VCP expression through Pim-1 via STAT3 is AR independent there by suggesting a role for VCP in CRPC. Transfection of LNCaP cells for VCP overexpression showed an increased cell proliferation, migration, and invasion where as its inhibition by NMS-873 showed the reverse effect causing cell death. Mechanistic studies demonstrate that cell death occurs due to apoptosis by endoplasmic reticulum (ER) stress, elevated cell cycle inhibitors p21, p27kip1, and active PARP and reduced Bcl-2. VCP promotes cell invasion and migration by altering E-cadherin and Vimentin levels inversely triggering EMT of PCa cells. VCP immunostaining revealed no staining in BPH but strong staining in PCa. This study determines VCP may play an important role in progression to CRPC and it can be a favorable target with to develop new therapies to treat ADT resistant prostate cancer.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is frequently upregulated in prostate cancer, and its overexpression conveys tumor growth and angiogenesis by metabolizing asymmetric dimethylarginine (ADMA).

Tissue microarray analysis confirmed higher dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression in prostate cancer (PCa) compared to benign and normal prostate tissues. DDAH1 regulates nitric oxide (NO) production by degrading endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA). This study examined whether DDAH1 has any physiological role in PCa progression. Using overexpression of DDAH1 in PCa (PC3 and LNCaP) cell lines, we found that DDAH1 promotes cell proliferation, migration and invasion by lowering ADMA levels, as well as increasing NO production. VEGF, HIF-1α and iNOS were upregulated in DDAH1 expressing cells as result of elevated NO. DDAH1 increased secretion of pro-angiogenic signals bFGF and IL-8, into conditioned media. Treatment of DDAH1-positive PCa cells with NOS inhibitors (L-NAME and 1400 W) attenuated DDAH1 activity to promote cell growth. Xenografts derived from these cells grew significantly faster (> twofold) than those derived from control cells. Proliferation rate of cells stably expressing mutant DDAH1 was same as control cells unlike wild-type DDAH1-positive PCa cells. Xenograft tumors derived from mutant-positive cells did not differ from control tumors. VEGF, HIF-1α and iNOS expression did not differ in DDAH1 mutant-positive tumors compared to control tumors, but was upregulated in wild-type DDAH1 overexpressing tumors. Furthermore, CD31 immunostaining on xenograft tissues demonstrated that DDAH1 tumors had high endothelial content than mutant DDAH1 tumors. These data suggest that DDAH1 is an important mediator of PCa progression and NO/DDAH pathway needs to be considered in developing therapeutic strategies targeted at PCa.

All-Trans-Retinoic Acid Stimulates Overexpression of Tumor Protein D52 (TPD52, Isoform 3) and Neuronal Differentiation of IMR-32 Cells.

Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration, and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (ß III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. J. Cell. Biochem. 118: 4358-4369, 2017. © 2017 Wiley Periodicals, Inc.

Prostate Cancer Associated Lipid Signatures in Serum Studied by ESI-Tandem Mass Spectrometryas Potential New Biomarkers.

Prostate cancer (PCa) is one amongst the most common cancersin western men. Incidence rate ofPCa is on the rise worldwide. The present study deals with theserum lipidome profiling of patients diagnosed with PCa to identify potential new biomarkers. We employed ESI-MS/MS and GC-MS for identification of significantly altered lipids in cancer patient's serum compared to controls. Lipidomic data revealed 24 lipids are significantly altered in cancer patinet's serum (n = 18) compared to normal (n = 18) with no history of PCa. By using hierarchical clustering and principal component analysis (PCA) we could clearly separate cancer patients from control group. Correlation and partition analysis along with Formal Concept Analysis (FCA) have identified that PC (39:6) and FA (22:3) could classify samples with higher certainty. Both the lipids, PC (39:6) and FA (22:3) could influence the cataloging of patients with 100% sensitivity (all 18 control samples are classified correctly) and 77.7% specificity (of 18 tumor samples 4 samples are misclassified) with p-value of 1.612×10-6 in Fischer's exact test. Further, we performed GC-MS to denote fatty acids altered in PCa patients and found that alpha-linolenic acid (ALA) levels are altered in PCa. We also performed an in vitro proliferation assay to determine the effect of ALA in survival of classical human PCa cell lines LNCaP and PC3. We hereby report that the altered lipids PC (39:6) and FA (22:3) offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis.

Prostate cancer-associated autoantibodies in serum against tumor-associated antigens as potential new biomarkers.

The limitations of the current prostate cancer (PCa) screening tests demands new biomarkers for early diagnosis of PCa. In this study, we aim to investigate serum autoantibody signatures as PCa specific biomarkers. PCa proteins were resolved by 2-DE and then transferred onto polyvinylidene difluoride membrane, which were subsequently incubated with either pooled serum from PCa patients or from normal controls. Mass spectrometry results have identified 18 antigens from 21 different 2-DE spots associated with PCa. Autoantibody response to antigens PRDX2, PRDX6 and ANXA11 in PCa patient's sera was confirmed using recombinant antigens. Further validation with an independent set of PCa patient's sera have shown relatively increased abundance of PRDX6 and ANXA11 antibodies in PCa patients. Formal concept analysis method was applied to assess whether the abundance of these autoantibodies could influence the classification of patients. However, sensitivity of the single antibody to discriminate prostate tumor and healthy controls varies from 70% to 80%, whereas combination of both PRDX6 and ANXA11 antibodies increased sensitivity to 90% for tumors and 100% for healthy controls. Therefore, we hereby report that the detection of these antibodies in PCa patient's serum in combination with the existing non-invasive diagnostic procedures may have significance in PCa diagnosis. BIOLOGICAL SIGNIFICANCE: The present study aimed to investigate serum autoantibody signatures as new biomarkers for early diagnosis of prostate cancer (PCa). To investigate serum autoantibodies in patients with PCa, we used proteomics approach based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Total tissue proteins extracted from prostate were separated by 2-DE and then transferred onto polyvinylidene difluoride (PVDF) membrane, which were subsequently incubated with either pooled serum from PCa patients or from normal controls with no history for PCa. Proteomic analysis results have identified 18 antigens that showed antibody response specifically to cancer patient's serum. For validation experiments using recombinant antigens, confirmed autoantibody response to three antigens PRDX2, PRDX6 and ANXA11. Further validation using a second independent set of PCa patient's sera has shown relatively increased abundance of PRDX6 and ANXA11 antibodies specifically in PCa patients. Partition analysis of patients based on abundance of autoantibodies highlighted a combination of both PRDX6 and ANXA11 antibodies in serum with 90% sensitivity in case of tumors and 100% in case of healthy controls. Therefore, we hereby report that the detection of these antibodies in PCa patient's serum in combination with known markers may have significance in diagnosis of PCa with further validation in larger cohort of samples.

Investigation of podophyllotoxin esters as potential anticancer agents: synthesis, biological studies and tubulin inhibition properties.

A series of fifteen podophyllotoxin derived esters have been synthesized and their anti-cancer properties have been evaluated against A549 (lung cancer), DU-145 (prostate cancer), HepG2 (liver cancer), HeLa (cervical cancer) and MCF-7 (breast cancer) cell lines. Five compounds of the series 8a, 8g-h, 8m and 8o showed IC50 values in the range of 0.71-10.94 µM. Among compounds, 8g and 8h showed significant cytotoxicity towards all the types of cancer studied. Cell cycle analysis revealed that the compounds 8a, 8m and 8o inhibit proliferation by cell cycle arrest. Also Hoechst-positive nucleus indicating apoptosis of these cells was observed in presence of 8g-h. Further studies revealed that these compounds inhibit tubulin polymerization and leads to the inactivation of AKT/PKB that are known to play an important role in the proliferation of cancer cells.