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The integrin β6 (ITGB6) gene, which encodes the limiting subunit of the integrin αvβ6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5′-RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region −24 to −18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located −289 to −150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis.

Although it is difficult to detect αvβ6 integrin (αvβ6) in normal epithelia cells, its expression is upregulated during wound healing and carcinogenesis. Overexpression of αvβ6 has been demonstrated in epithelial cell carcinomas, such as adenocarcinoma of the colon and ovary. However, the expression of αvβ6 has not been reported in hepatocellular carcinoma (HCC). We previously indicated that LPA may induce αvβ6-mediated TGF-β1 signaling mechanisms during the pathogenesis of lung injury and fibrosis. In addition, transforming growth factor-β1 (TGF-β1) and lysophosphatidic acid (LPA) have been demonstrated to participate in the progression of HCC. In the present study, we hypothesized that TGF-β1 and LPA would serve a key role in the subunit integrin β6 (Itgβ6) transcriptional regulatory mechanism in HCC. It was identified that human HCC tissues and Hep-3B cells expressed Itgβ6. Treatment of Hep-3B with TGF-β1 or LPA increased the expression of Itgβ6. Furthermore, truncation experiments indicated a positive regulatory region at -326 to -157 bp of the Itgβ6 promoter. TGF-β1 and LPA increased transcriptional activation at this regulatory region. To the best of our knowledge, the present study was the first to demonstrate Itgβ6 expression in HCC, and the data indicate that TGF-β1 and LPA regulate Itgβ6 expression through the Itgβ6 gene promoter, which is an important factor in the development of HCC.

Adoptive transfer of tumor-specific T cell receptor (TCR)-engineered T cells, TCR-T therapy, has shown great efficacy in clinical trials against solid tumors. A cervical cancer patient with bone metastasis was treated with a cell-based immunotherapy, named Multiple-Antigens Stimulating Cellular Therapy (MASCT), which is a combination of multiple tumor antigen peptides loaded dendritic cells (DCs) and autologous T cells stimulated by these DCs. After repeated treatment, the patient showed partially response and remained stable disease for 20 months. Moreover, boosted specific immune responses were detected in patient's periphery blood by IFNγ-Elispot assay against various tumor antigens, such as CEA, RGS5 and HPV18/58. The clinical benefits of this patient indicated that tumor-specific T cells were expanded in vivo and played an important role to control tumor progression. These T cells may be the good sources to isolate tumor-specific TCRs for TCR-T therapies. Given that, patient's PBMCs were stimulated in vitro with selected tumor peptides respectively, such as CEA, RGS5 and HPV18 E7. Tumor-specific T cells were defined as IFNγ-secreting T cells upon peptide stimulation and were further enriched by using magnetic beads. Part of the enriched T cells were analyzed for TCR alpha/beta repertoire via NGS, the other part were sorted into single T cells and analyzed for TCR alpha/beta pairing. The TCR repertoire NGS data revealed that the tumor-specific T cells harbor several hundred unique TCR alpha/beta clonotypes. Some unique TCR alpha/beta clonotypes were considered as tumor-specific TCR candidates, since the copy numbersof these clonotypes were significantly and repeatedly increased after stimulation with CEA, RGS5 or HPV18 E7, and further increased after IFNγ enrichment. After TCR alpha/beta pairing confirmation by single T cell sequencing, alpha and beta chains of these TCRs were sent to synthesis for further investigation. In conclusion we have successfully identified specific TCR clonotypes targeting selected tumor antigens from a MASCT-benefited cervical cancer patient. After functional validations, including epitope specificity, HLA restriction as well as tumor recognition, these tumor-specific TCRs may be good candidates for developing safer and more effective TCR-T therapy targeting multiple tumor antigens. Citation Format: Xiaoling Liang, Shudan Ou, Minjun Ma, Xihe Chen, Xiangjun Zhou, Yanyan Han. To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2554.

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