We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase (1). Here we show that CDA1-induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21Waf1/Cip1 (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time-and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53-and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-β (TGF-β) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-β signaling, modulating the expression of TGF-β, TGF-β receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-β together were synergistic in stimulating TGF-β signaling and target gene expression. CDA1 knockdown effectively blocked TGF-β-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-β type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-β signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-β signaling in the kidney.

CDA1 plays an important role in vascular ECM accumulation by amplifying TGF-beta signalling. This is critical for the profibrotic effect of TGF-beta in the vasculature. CDA1 is therefore a potential target for attenuating vascular ECM accumulation caused by enhanced TGF-beta action, as seen in diabetic atherosclerosis.

Cell division autoantigen 1 (CDA1) enhances TGF-b signaling in renal and vascular cells, and renal expression of CDA1 is elevated in animal models of diabetes. In this study, we investigated the genetic deletion of Tspyl2, the gene encoding CDA1, in C57BL6 and ApoE knockout mice. The increased renal expression of TGF-b1, TGF-b type I and II receptors, and phosphorylated Smad3 associated with diabetes in wild-type mice was attenuated in diabetic CDA1 knockout mice. Notably, CDA1 deletion significantly reduced diabetes-associated renal matrix accumulation and immunohistochemical staining for collagens III and IV and attenuated glomerular and tubulointerstitial injury indices, despite the presence of persistent hyperglycemia, polyuria, renal hypertrophy, and hyperfiltration. Furthermore, CDA1 deletion reduced gene expression of TGF-b1 receptors in the kidney, resulting in a functionally attenuated response to exogenous TGF-b, including reduced levels of phosphorylated Smad3 and ERK1/2, in primary kidney cells from CDA1 knockout animals. Taken together, these data suggest that CDA1 deletion reduces but does not block renal TGF-b signaling. Because direct antagonism of TGF-b or its receptors has unwanted effects, CDA1 may be a potential therapeutic target for retarding DN and perhaps, other kidney diseases associated with TGF-b-mediated fibrogenesis.

Current studies aiming at identifying single immune markers with prognostic value have limitations in the context of complex antitumor immunity and cancer immune evasion. Here, we show how the integration of several immune markers influences the predictions of prognosis of gastric cancer (GC) patients. We analyzed Tissue Microarray (TMA) by multiplex immunohistochemistry and measured the expression of immune checkpoint molecule PD-L1 together with antitumor CD8 T cells and immune suppressive FOXP3 Treg cells in a cohort of GC patients. Unsupervised hierarchical clustering analysis of these markers was used to define correlations between CD8 T, FOXP3 Treg and PD-L1 cell densities. We found that FOXP3 and PD-L1 densities were elevated while CD8 T cells were decreased in tumor tissues compared to their adjacent normal tissues. However, patient stratification based on each one of these markers individually did not show significant prognostic value on patient survival. Conversely, combination of the ratios of CD8/FOXP3 and CD8/PD-L1 enabled the identification of patient subgroups with different survival outcomes. As such, high densities of PD-L1 in patients with high CD8/FOXP3 and low CD8/PD-L1 ratios correlated with increased survival. Collectively, this work demonstrates the need for the integration of several immune markers to obtain more meaningful survival prognosis and patient stratification. In addition, our work provides insights into the complex tumor immune evasion and immune regulation by the tumor-infiltrating effector and suppressor cells, informing on the best use of immunotherapy options for treating patients.