Infiltrating T cells promote renal cell carcinoma (RCC) progression via altering the estrogen receptor β-DAB2IP signals

Abstract

Previous studies indicated the T cells, one of the most common types of immune cells existing in the microenvironment of renal cell carcinoma (RCC), may influence the progression of RCC. The potential linkage of T cells and the estrogen receptor beta (ERβ), a key player to impact RCC progression, however, remains unclear. Our results demonstrate that RCC cells can recruit more T cells than non-malignant kidney cells. Using an in vitro matrigel invasion system, we found infiltrating T cells could promote RCC cells invasion via increasing ERβ expression and transcriptional activity. Mechanism dissection suggested that co-culturing T cells with RCC cells released more T cell attraction factors, including IFN-γ, CCL3 and CCL5, suggesting a positive regulatory feed-back mechanism. Meanwhile, infiltrating T cells may also promote RCC cell invasion via increased ERβ and decreased DAB2IP expressions, and knocking down DAB2IP can then reverse the T cells-promoted RCC cell invasion. Together, our results suggest that infiltrating T cells may promote RCC cell invasion via increasing the RCC cell ERβ expression to inhibit the tumor suppressor DAB2IP signals. Further mechanism dissection showed that co-culturing T cells with RCC cells could produce more IGF-1 and FGF-7, which may enhance the ERβ transcriptional activity. The newly identified relationship between infiltrating T cells/ERβ/DAB2IP signals may provide a novel therapeutic target in the development of agents against RCC.

Keywords: CD4+ T cells; DAB2IP; ERβ; RCC; metastasis.

Figure 1. RCC cells can better attract CD4+ T cells than the non-malignant kidney cells

Transwell migration assay was applied to study the capability of RCC cell to attract T cells. Non-malignant kidney cells (HKC2) and RCC cells (786-O and A498) were seeded in the lower chamber of transwells, and T cells were placed in the upper chambers with 5 μm pore size members. After 6 hrs of incubation, the media in the bottom well was collected and numbers of migrated T cells were counted by BioRed T20 cell counting system. Each experiment was independently repeated three times, each time in triplicate. *p < 0.05 vs HKC-2.

Figure 2. Recruited T cells could promote RCC cells invasion

RCC cells, 786-O and A498, were cultured alone or co-cultured with T cells for 48 hrs. In a transwell system, the RCC cells with/without being co-cultured were re-seeded into the inserted wells that were pre-coated with matrigel for invasion assay. Invaded cells were stained by 1% toluidine blue (upper panels) and quantifications of invaded cells are presented in bottom panel. Each experiment was independently repeated three times, each time in triplicate. *p < 0.05 vs RCC alone.

Figure 3. Co-culture of RCC and CD4+ T cells (HH) can activate ERβ transcriptional activity and increase ERβ expression in RCC cells

A. ERβ transcriptional activity was increased by the conditioned media (CM) collected from co-cultured cells. 293T cells were transfected with ERβ, and (ERE)₃-luciferase reporter plasmid by lipofetamine 3000 following the manufacture's protocol. CM was collected from RCC alone, T cells alone, or RCC+T cells co-culture and was used to treat 239T cells after 48 hrs transfection with ERβ and (ERE)₃-luciferase reporter or control plasmids. 10 nM E2 treatment was applied as positive control for induction of ERβ activity. *p < 0.05 vs vector. B. Co-culture with T cells could increase ERβ protein expression in RCC cells. ERβ protein was detected by anti-ERβ polyclonal antibody (GTX 110607, GeneTex) at 1 to 1000 dilution in 1 × PBS with 5% BSA. *, #, § p < 0.05; * vs shLuc without T cell co-culture; # vs shLuc with T cells; § vs shERβ without T cell co-culture. C. T cells co-culture-induced ERβ can enhance RCC cell invasion. We introduced shERβ or shLuc in RCC cells, and then RCC cells were co-cultured with T cells. After incubating 48 hrs, RCC cells were re-seeded into upper wells of a transwell system to perform the matrigel invasion assay. * vs shLuc without T cell co-culture; # vs shLuc with T cell co-culture; *, # p < 0.05. D. Increased ERβ plays important roles in the T cells-enhanced RCC invasion. ER antagonist, ICI182,780 (1 μM), or mock control was used to treat 786-O cells for 24 hrs. Those 786-O cells were either cultured alone, or co-cultured with T cells for 48 hrs. RCC Cells were then seeded in the matrigel pre-coated insert wells for the transwell invasion assay. Each experiment was independently repeated three times, each time in triplicate. *, #, § p < 0.05 * vs 786-O no treatment; # vs 786-O+T cell no treatment T cell; § vs 786-O with ICI treatment

Figure 4. Recruited T cells can promote RCC cell invasion through ERβ/DAB2IP signal pathway

A–B. Co-culture of RCC cells with T cells can increase ERβ, but decrease DAB2IP expression, in both 786-O and A498 cells. Total cell lysates were collected from RCC cells cultured alone or co-cultured with T cells for 48 hr. 50 μg of total protein was loaded in each well, anti-ERβ and anti-DAB2IP antibodies were used to detect their protein levels. *p < 0.05. C. ERβ could inhibit DAB2IP protein expression level in RCC. Total cell lysates were collected from RCC cells cultured alone or with T cells. DAB2IP expression was compared between these two conditions (left panels). ERβ could inhibit DAB2IP expression (right panels). DAB2IP protein level was compared between 786-O/sh control (shLuc) and 786-O/shERβ. D. ERβ-induced RCC invasion may function through down-regulation of DAB2IP. Lentiviral shERβ or shLuciferase (shLuc) was used to study the ERβ knockdown effects on RCC cell invasion. Our data showed that shERβ could reduce the RCC cell invasion. As ERβ knockdown can lead to the increased DAB2IP, we next tested whether knockdown of DAP2IP could reverse the ERβ knockdown-reduced RCC invasion. Lentiviral shDAB2IP or scramble control (shcon) were transduced into 786-O cells. 786-O cells express a high endogenous ERβ, thus 786-O cells are used to knock down ERβ for functional assay. After lentiviral transduction of different shRNA and controls, puromycin selection was used to enrich the transduced 786-O cells. Cells were then co-cultured with/without T cells, and then re-seeded to a new transwell matrigel invasion system to study the roles of ERβ and DAB2IP in 786-O cells invasion. The invaded 786-O cells were stained with 1% toluidine B in PBS (bottom panel). Quantifications of invaded 786-O cells were shown in right panel. *, #, § and $ p < 0.05 * vs shLuc/sh cp mom 786-O; # shERβ/shcon in 786-O; § vs shLuc/shcon in 786-O+T cells; $ vs shERβ/shcon in 786-O+T cells.

Figure 5. RCC cells promote T cells to express more IGF, FGF and IFN-γ, which could then activate the ERβ pathway in RCC cells

RCC and T cells were co-cultured for 48 hrs, the RCC or T cells were separated, and then metastasis-related gene expressions were analyzed by Q-PCR. A. RCC cells (with T cells) vs RCC cells (only). *p < 0.05. B. T cells (with RCC co-culture) vs T cells (only). C. ERβ in RCC could affect T cells activity. RCC cells with/without ERβ knockdown cultured alone, or co-cultured with T cells. After 48 hrs incubation, target genes were analyzed in T cells. *, # p < 0.05 * vs T cell alone; # vs T cell+786-O shLuc

Figure 6. Co-culture of RCC cells with T cells can enhance IFN-γ and IGF production in T cells via increasing ERβ in T cells

T cells transduced with lentiviral ERβ or shLuc were subjected to puromycin selection to establish the stable clones in T cells. A. T cells, with shERβ or with shLuc, were co-cultured with 786-O cells to test the invasion capability. * P < 0.05, *vs 786-O cells only. B. ERβ, IFN-γ and IGF-1 gene expression in T cells (shLuc, shERβ) alone and co-cultured with RCC cells. Each experiment was independently repeated 3 times. *, # p < 0.05, *vs T cells shLuc alone; # vs T cell shLuc+786-O; p ≤ 0.05 was considered statistically significant.

Figure 7. Schematic presentation of the interaction mechanisms of T cells with RCC cells in the tumor microenvironment

After T cells migrated toward RCC cells, both ERβ transcriptional activity and protein expression increase, but DAB2IP expression decreases in RCC cells. In addition to ERβ, IFN-γ, CCL3, CCL5, IGF and FGF7 also increased in both RCC and T cells after co-culture. IFN-γ and CCL3 and CCL5 may correlate with T cell recruitment. In addition to E2, IGF and FGF7 have been demonstrated to activate ERs transactivation. All these increased genes were regulated by ERβ when the infiltrated T cells interact with RCC cells in the tumor microenvironment.