Endogenous Retroviral-K Envelope Is a Novel Tumor Antigen and Prognostic Indicator of Renal Cell Carcinoma

Abstract

Renal cell carcinoma (RCC) is one of the ten most common cancers for men and women with an approximate 75% overall 5-year survival. Sixteen histological tumor subtypes exist and the most common are papillary, chromophobe and clear cell renal cell carcinoma (ccRCC) representing 85% of all RCC. Although epigenetically silenced, endogenous retroviral (ERV) genes become activated in tumors and function to ignite immune responses. Research has intensified to understand ERV protein function and their role as tumor antigens and targets for cancer (immune) therapy. ERV-K env is overexpressed and implicated as a therapeutic target for breast cancer, however studies in RCC are limited. In this investigation a human RCC tissue microarray (TMA) (n=374) predominantly consisting of the most common histological tumor subtypes was hybridized with an ERV-K env antibody and correlated with patient clinical data. TMA results showed the highest amount of ERV-K env protein expression and the strongest significant membrane expression in ccRCC versus other RCC subtypes. High ERV-K env total protein expression of all tumor subtypes significantly correlated with low tumor grading and a longer disease specific survival using multivariable analyses. Cell proliferation and invasion were assayed using the kidney cell lines HEK293 with wild-type p53 and a ccRCC cell line MZ1257RC mutated for p53. Transfecting these cell lines with a codon optimized ERV-K113 env overexpressing CMV vector was performed with or without 5'-Aza-2'-deoxycytidine (Aza) treatment to sustain promoter de-methylation. MZ1257RC showed induction of ERV-K113 expression and significantly increased both proliferation and invasion in the presence or absence of Aza. HEK293 cells demonstrated a restriction of ERV-K113 env expression and invasion with no changes in proliferation in the absence of Aza. However, in the presence of Aza despite increased ERV-K113 env expression, an inhibition of HEK293 proliferation and a further restriction of invasion was found. This study supports ERV-K env as a single prognostic indicator for better survival of RCC, which we propose represents a new tumor antigen. In addition, ERV-K env significantly regulates proliferation and invasion depending on p53 status and Aza treatment.

Keywords: ERV-K; azacytidine; endogenous retrovirus; invasion; p53; patient prognosis; renal cell carcinoma; tumor antigen.

Figure 1

ERV gene expression in primary tissues. (A) ERV expression in ccRCC and control kidney tissues. Seven different ERV gene families were analyzed for expression in molecules/ng RNA (log10) (Y-axis) from 14 primary ccRCC and 11 patient matched tumor associated control tissues (X-axis). Gene expression was then compared using Mann-Whitney two-tailed test, where all ccRCC tumors were significantly increased compared to controls (p < 0.0001). (B) ERV6q15 env expression ratios in primary ccRCC. The graph shows for each ccRCC (n= 14; same tissues as in A) the specific ERV expression from 7 families (A) in a ratio (log10) (Y-axis) compared with ERV-E6q15 (X-axis - E6q15).

Figure 2

Representative microscopic images of TMA samples of different RCC tumor subtypes hybridized with a specific ERV-K env antibody. (A) Example of a ccRCC (Tumor #21 TMA I) with strong protein expression of ERV-K env at the cell membrane. (B) Example of a ccRCC (Tumor #23 TMA I) with strong cytosolic enrichment for ERV-K env protein. (C) Example of a RCC papillary tumor with strong cytosolic enrichment for ERV-K env protein (Tumor #28 TMA II). (D) Example of a ccRCC with sarcomatoid features (Tumor #20 TMA III) with strong membranous as well as a partly cytosolic ERV-K env staining. Bar indicates 50µm.

Figure 3

ERV-K env protein localization and clinical correlations between RCC tumor subtypes. (A) ERV-K env cytosolic (cyto) or membrane (mem) localization represented as H-scores (Y-axis; 0 - 400) for each RCC tumor TMA subtype (X-axis). TMA; ccRCC = clear cell carcinoma (n= 288) with sarcomatoid (Sarcomat) features (n= 8); Chromo = chromophobe (n= 27); papill-1 and -2 = Papillary 1 (n= 18) and 2 (n= 27). ***p< 0.0001; **p= 0.0003, ns = not significant. Red bar crosses represent the mean for each cohort tumor subtype. (B) Bar graphs comparing ERV-K env membranous or (C) cytosolic staining or (D) total protein expression (combined) with the H-score significance and RCC tumor subtypes. (E) Bar graphs comparing ERV-K env membranous or (F) cytosolic or (G) total protein expression (combined) for each RCC tumor subtype staining with the H-score significance comparing RCC tumor subtypes and tumor grading (G1, G2, G3). All p-values are indicated within the graphs or ***p< 0.001.

Figure 4

ERV-K env and associations with RCC patient survival. (A) Kaplan-Meier survival curves {Disease specific survival (DSS) in months} of all RCC patients as represented from the TMA showing the association of ERV-K env membranous, cytosolic and total protein expression (combined) according to the H-score (> blue or < red). Note that ERV-K env total protein shows a significance for longer DSS survival. (B) Kaplan-Meier survival curves (Disease specific survival in months) of ccRCC patients represented from the TMA showing the association of ERV-K env membranous, cytosolic and total protein expression (combined). (C) Kaplan-Meier survival curves of all RCC patients represented from the TMA according to staging and grading. For staging the following regressions were: pT2 vs pT1: HR 13.7; p= 0.0002; 95%-CI: 3.39 - 55.33; pT3 vs pT1: HR 6.36; p= 0.03; 95%-CI: 1.17 - 34.71; pT3 vs pT2: HR 0.46; p= 0.32; 95%-CI: 0.1 – 2.08. For Grading the regressions were: G3 vs G2 + G1: HR 2.28; p= 0.23; 95%-CI: 0.6 - 8.71. For all curves log rank p-values are shown as well as multivariable adjusted Hazards ratios (HR) calculated by using Cox regression models. Partitioning tests (mono-forest prediction) were performed to determine “cut-off” levels for each tumor subtype because of known biological differences between the histological RCC tumor subtypes.

Figure 5

ERV gene expression in ccRCC cell lines and functional studies. (A) Graph shows gene expression (molecules/ng RNA, Y-axis) profiling 16 different ERV gene families (X-axis) of 12 different cell lines (ccRCC = 11 see Materials and Methods; Red= MZ1257RC ccRCC cell line; Green = HEK293). Note that ERV-Fc1 env and ERV-Rb env are not shown on the graph since they were undetectable for gene expression. (B) Graph shows MZ1257RC cell proliferation (Y-axis = total cell counts) following transient transfection with the overexpressing CMV vector containing a codon optimized ERV-K113 env gene at 44 h, 68 h and 92 h post transfection in the presence or absence of Aza. (n= 4); *p = 0.0286. (C) Graph shows MZ1257RC 3D cell invasion into collagen (Y-axis = # of invaded cells/mm²) following transient transfection with the overexpressing CMV vector containing a codon optimized ERV-K113 env gene at 68 h post transfection in the presence or absence of Aza. (n= 6), ***p < 0.0001; ns = non-significant. (D) Graph shows HEK293 3D invasion into collagen (Y-axis = # of invaded cells/mm²) following transient transfection with the overexpressing CMV vector containing a codon optimized ERV-K113 env gene at 68 h post transfection in the presence or absence of Aza. (n= 3), ***p < 0.0001.

Figure 6

Results and model showing transcriptional regulations and cellular functions of HEK293 and MZ1257RC cell lines following ERV-K113 env gene transfection in the presence or absence of Aza treatment. Left schematic shows top HEK293 cells or bottom MZ1257RC cells transfected with the overexpressing CMV vector containing a codon optimized ERV-K113 env gene in the absence of Aza (-Aza). The CMV promoter (grey) with p53 binding to TATA binding protein (TBP) (38) is methylated (Me). Note that the transcription factor SP1 is not bound. The p53 wt protein is a repressor (green) for HEK293 cells, but mutant (dysfunctional) p53 (red) for the MZ1257RC cell line. For HEK293 cells the green arrow (ERV-K env) indicates less expression, when compared with MZ1257RC cells. A 2.99-fold increase of ERV-K113 env gene expression for MZ1257RC (2^-ΔΔCt = -Aza = 3,113.2) is indicated compared to HEK293 cells (2^-ΔΔCt = -Aza = 1,038.5) at 24 h post transfection. Right schematic shows top HEK293 cells or bottom MZ1257RC cells transfected with the overexpressing CMV vector containing a codon optimized ERV-K113 env gene in the presence of Aza (+Aza). P53, TBP and SP1 (40) are bound at a de-methylated CMV promoter (grey) promoting transcription. In the presence of Aza an inhibition of DNA-methylation (Me) and a lack of p53 repression is predicted for HEK293 cells. For both cell lines ERV-K113 env green arrows indicate higher levels of expression when compared to –Aza treated cells (left schematic). ERV-K113 env gene expression for HEK293 was 7.12-fold higher (2^-ΔΔCt = +Aza = 7398.7) and for MZ1257RC cells a 2.86-fold induction (2^-ΔΔCt = +Aza = 8927.69) at 24 h. Note that for both cell lines the 2^-ΔΔCt ERV-K113 env gene expression levels were similar at 24 h after Aza treatments. To the far right for both cell lines functional outcomes are indicated for proliferation (at 44 h, 68 h, 92 h) and invasion (68 h), when compared to in the presence or absence of Aza treatment (see Figure 5 , Supplementary Figure 2 ) (black arrows = no change; red arrows = inhibition; green arrows = increase).