SQAP, an acyl sulfoquinovosyl derivative, suppresses expression of histone deacetylase and induces cell death of cancer cells under hypoxic conditions.

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.

Upstream Position of Proline Defines Peptide-HLA Class I Repertoire Formation and CD8+ T Cell Responses.

Cytotoxic CD8+ T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.

The Antigen ASB4 on Cancer Stem Cells Serves as a Target for CTL Immunotherapy of Colorectal Cancer.

Colorectal cancer consists of a small number of cancer stem cells (CSC) and many non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because they survive conventional chemo- and radiotherapies and perpetuate tumor formation in vivo In this study, we conducted an HLA ligandome analysis to survey HLA-A24 peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified an antigen, ASB4, which was processed and presented by a CSC subset but not by non-CSCs. The ASB4 gene was expressed in CSCs of colorectal cancer, but not in cells that had differentiated into non-CSCs. Because ASB4 was not expressed by normal tissues, its peptide epitope elicited CD8+ cytotoxic T-cell (CTL) responses, which lysed CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-associated antigen that can elicit CTL responses specific to CSCs and can discriminate between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and effectively prevented tumor growth in a mouse model. As the cancer cells implanted in these mice contained very few CSCs, the elimination of a CSC subset could be the condition necessary and sufficient to control tumor formation in vivo These results suggest that CTL-based immunotherapies against colorectal CSCs might be useful for preventing relapses. Cancer Immunol Res; 6(3); 358-69. ©2018 AACR.

Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigen-specific CTL recognition.

Cytotoxic T-lymphocytes (CTLs) lyse target cells after recognizing the complexes of peptides and MHC class I molecules (pMHC I) on cell surfaces. Tapasin is an essential component of the peptide-loading complex (PLC) and its absence influences the surface repertoire of MHC class I peptides. In the present study, we assessed tapasin expression in 85 primary tumor lesions of non-small cell lung cancer (NSCLC) patients, demonstrating that tapasin expression positively correlated with patient survival. CD8+ T-cell infiltration of tumor lesions was synergistically observed with tapasin expression and correlated positively with survival. To establish a direct link between loss of tapasin and CTL recognition in human cancer models, we targeted the tapasin gene by CRISPR/Cas9 system and generated tapasin-deficient variants of human lung as well as colon cancer cells. We induced the CTLs recognizing endogenous tumor-associated antigens (TAA), survivin or cep55, and they responded to each tapasin-proficient wild type. In contrast, both CTL lines ignored the tapasin-deficient variants despite their antigen expression. Moreover, the adoptive transfer of the cep55-specific CTL line failed to prevent tumor growth in mice bearing the tapasin-deficient variant. Loss of tapasin most likely limited antigen processing of TAAs and led to escape from TAA-specific CTL recognition. Tapasin expression is thus a key for CTL surveillance against human cancers.

Anti-angiogenesis effect of 3'-sulfoquinovosyl-1'-monoacylglycerol via upregulation of thrombospondin 1.

We previously reported that 3'-sulfoquinovosyl-1'-monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti-angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real-time polymerase chain reaction. Consequently, upregulation of thrombospondin 1 (TSP-1) in SQMG-treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP-1 upregulation by SQMG, we established stable TSP-1-knockdown transformants (TSP1-KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti-tumor assay. On the reporter assay, transcriptional upregulation of TSP-1 in TSP1-KT could not be induced by SQMG, thus suggesting that TSP-1 upregulation by SQMG occurred via TSP-1 molecule. In addition, growth of TSP1-KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation.