GTF2IRD1 overexpression promotes tumor progression and correlates with less CD8+ T cells infiltration in pancreatic cancer

Abstract Background: General Transcription Factor II-I Repeat Domain-Containing Protein 1 (GTF2IRD1) is a member of the GTF21 gene family, which encodes a set of multifunctional transcription factors. However, the potential function of GTF2IRD1 in pancreatic cancer (PC) still remains unknown. Study on GTF2IRD1 might provide a new insight into the carcinogenesis and therapeutics of PC. Methods: In the current study, the clinical significance and potential biological of GTF2IRD1 were evaluated by bioinformatics analysis. The oncogenic role of GTF2IRD1 in PC was also determined using in vitro studies. Possible associations between GTF2IRD1 expression and tumor immunity were analyzed using ESTIMATE algorithm and single-sample Gene Set Enrichment Analysis (ssGSEA). Results: GTF2IRD1 expression was significantly up-regulated in tumor tissues, and positively associated with higher histologic grade, higher American Joint Committee on Cancer (AJCC) stage, and worse prognosis. Function enrichment analysis demonstrated that GTF2IRD1 may be involved in pancreatic adenocarcinoma pathway, TGF-β signaling pathway, and tumor-infiltrating lymphocyte (TIL) related biological functions, such as T-cell receptor signaling pathway, leukocyte transendothelial migration, resistin as a regulator of inflammation, and regulation of leukocyte-mediated cytotoxicity. Knockdown of GTF2IRD1 expression inhibited cancer cell proliferation, colony formation, and invasion in vitro. ESTIMATE algorithm and ssGSEA demonstrated that GTF2IRD1 expression negatively correlated with the infiltration and anti-tumor activity of TILs, especially for CD8+ T cells. Conclusion: The study demonstrates that GTF2IRD1 overexpression promotes tumor progression and correlates with less CD8+ T cells infiltration in PC.


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As one of the most aggressive malignancies, pancreatic cancer (PC) causes 4.5% of 55 all cancer-related deaths worldwide in 2015 [1]. PC is widely recognized as a "cold" 56 tumor due to its notable immunosuppression [2]; thus, enhancing or recruiting 57 anti-tumor immune cells into the tumor microenvironment (TME) is a promising 58 therapeutic strategy for PC [3]. In recent years, immune checkpoint inhibitors (ICIs) 59 have been effectively used for several solid tumors, such as melanoma, non-small cell 60 lung cancer, and hepatocellular carcinoma [4][5][6]. However, these drugs fail to be 61 effective treatments for patients with advanced PC due to the lack of CD8 + T cells in 62 the TME of PC [7]. Therefore, exploring the molecular mechanisms underlying PC 63 progression and immune suppression is critical for developing more effective 64 immunotherapies to improve survival rates.
General Transcription Factor II-I Repeat Domain-Containing Protein 1 (GTF2IRD1) 66 locating at 7q11.23, is a member of the GTF21 gene family, which encodes a set of 67 multifunctional transcription factors [8]. Early studies on GTF2IRD1 mainly focused 68 on its association with Williams-Beuren syndrome, which is a genetic disorder 69 associated with multiple systemic abnormalities, including hypertension, diabetes, 70 osteoporosis, and anxiety [9,10]. Previous oncology studies about GTF2IRD1 have 71 been conducted in only three cancers, including colorectal cancer, breast cancer, and 72 retinoblastoma [8,11]. Nevertheless, the precise function and mechanism of 73 GTF2IRD1 in PC has not been studied to date. Besides, the potential association 74 between GTF2IRD1 expression and immune infiltration in PC still remain to be 75 elucidated. 76 In the present study, for the first time, we comprehensively analyzed the expression of 77 GTF2IRD1 and its correlation with prognosis in PC. Then, we also analyzed the 78 potential underlying biological role of GTF2IRD1 in PC using functional enrichment 79 analysis in ConsensuspathDB (http://cpdb.molgen.mpg.de/). And the oncogenic role 80 of GTF2IRD1 in PC was determined using in vitro studies. In addition, potential 81 association between the expression of GTF2IRD1 and immune cells infiltration levels 82 in PC was explored using ESTIMATE algorithm and single sample Gene Set 83 Enrichment Analysis (ssGSEA).

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Data acquisition 86 First, the expression level of GTF2IRD1 in PC was analyzed using the Gene Corning, NY, USA). Briefly, the transfected cells were allowed to grow to confluence.

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Cells (10 5 ) were suspended in 100 μL serum-free medium and plated in triplicates Corp., Tokyo, Japan) and nine visual fields were observed in each group, respectively.

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Using R package estimate, ESTIMATE algorithm was conducted to assess the level of coefficients. The correlation between GTF2IRD1 expression and CD8 + T cells was 190 also analyzed using the TIMER web tool (https://cistrome.shinyapps.io/timer/).

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Furthermore, to further confirm the association between GTF2IRD1 and immune 192 infiltration in PC, we conducted correlation analysis between GTF2IRD1 and the 193 marker genes from TILs using the GEPIA database.

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First, the GEPIA database demonstrated that the expression of GTF2IRD1 was 197 significantly higher in PC tissues than that in normal pancreatic tissues (P value <  = -0.58, P < 0.05 for T cell migration; Cor = -0.47, P < 0.05 for regulation of 238 leukocyte mediated cytotoxicity) (Fig. 2). Furthermore, pathway enrichment analysis 239 showed that GTF2IRD1 may be involved in pancreatic adenocarcinoma pathway,

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suggested that GTF2IRD1 overexpression provided necessary support for 248 tumorigenesis, invasion, and the regulation of TILs infiltration in PC. To further evaluate the functional role of GTF2IRD1 in tumor cells proliferation and 251 invasion, we transfected PANC-1 and AsPC-1 cells with si-GTF2IRD1. Abrogated 252 levels of GTF2IRD1 in these two cells were validated using RT-qPCR analysis 253 (p<0.001) (Fig. 3A). Using the MTT assay, we found that cell proliferation was 254 significantly inhibited in GTF2IRD1-depleted PANC-1 and AsPC-1 cells (p<0.0001) 255 (Fig. 3B). In the plate colony formation assay, the number of colonies in the 256 si-GTF2IRD1 group was significantly lower than that in the NC (control) group 257 (p<0.0001) (Fig. 3C). Moreover, the transwell assay performed to determine the 258 invasive capacity of PANC-1 and AsPC-1 cells transfected with si-GTF2IRD1 259 revealed that the number of invasion cells was significantly less in the si-GTF2IRD1 260 group than that in the NC group (p<0.0001) (Fig. 3D). These findings suggest 261 GTF2IRD1 is necessary for PC cell proliferation and invasion.

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Our study provides new insights into the tumor immune microenvironment and 349 immune-based therapies for PC. We To the best of our knowledge, the current study is 350 the first to provide clear evidence for the association between GTF2IRD1 expression 351 and CD8 + T cells infiltration in PC. However, some limitations exist in this study.

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Firstly, this is a retrospective study based on publicly available datasets. Therefore, 353 the quality of data can influence the study outcomes. Secondly, the potential role of 354 GTF2IRD1 in pancreatic carcinogenesis was validated in vitro, but not in vivo.

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Thirdly, further experimental studies should be performed to validate the potential In summary, our study identified GTF2IRD1 as an unfavorable prognostic biomarker.

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And GTF2IRD1 overexpression promotes tumor progression and correlates with less 362 CD8 + T cells infiltration, which might be a novel therapeutic target in PC.

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Availability of data and material 368 All data used and analyzed in this study is available from the correspondence upon 369 reasonable request. 371 We declare that we have no conflict of interest.