The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

Abstract

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF–DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer. Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF–DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer. Development of clear cell renal cell carcinoma (ccRCC) is characterized by loss of function of the von Hippel-Lindau (VHL) tumor suppressor protein (1Gossage L. Eisen T. Maher E.R. VHL, the story of a tumour suppressor gene.Nat. Rev. Cancer. 2015; 15: 55-64Google Scholar). Advanced tumors harbor several additional somatic mutations including frequent deletions of genes coding for chromatin modifying enzymes, but timing the order of events has pinpointed loss of VHL as the truncal mutation in ccRCC (2Mitchell T.J. Turajlic S. Rowan A. Nicol D. Farmery J.H.R. O'Brien T. Martincorena I. Tarpey P. Angelopoulos N. Yates L.R. Butler A.P. Raine K. Stewart G.D. Challacombe B. Fernando A. et al.Timing the landmark events in the evolution of clear cell renal cell cancer: TRACERx renal.Cell. 2018; 173: 611-623.e17Google Scholar, 3Varela I. Tarpey P. Raine K. Huang D. Ong C.K. Stephens P. Davies H. Jones D. Lin M.L. Teague J. Bignell G. Butler A. Cho J. Dalgliesh G.L. Galappaththige D. et al.Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma.Nature. 2011; 469: 539-542Google Scholar, 4Cancer Genome Atlas Research NetworkComprehensive molecular characterization of clear cell renal cell carcinoma.Nature. 2013; 499: 43-49Google Scholar). Dysfunction of VHL leads to stabilization of the alpha subunits of hypoxia-inducible transcription factors (HIF). This can be observed already in early cancerous lesions in the kidney and triggers a broad pseudo hypoxic transcriptional program (5Mandriota S.J. Turner K.J. Davies D.R. Murray P.G. Morgan N.V. Sowter H.M. Wykoff C.C. Maher E.R. Harris A.L. Ratcliffe P.J. Maxwell P.H. HIF activation identifies early lesions in VHL kidneys: Evidence for site-specific tumor suppressor function in the nephron.Cancer Cell. 2002; 1: 459-468Google Scholar). The function of HIF in ccRCC has been well studied, but several aspects are still under debate. For example, data from genetic analyses and xenograft studies suggest that HIF-1α slows tumor progression, whereas HIF-2α may promote tumor growth (6Shen C. Beroukhim R. Schumacher S.E. Zhou J. Chang M. Signoretti S. Kaelin Jr., W.G. Genetic and functional studies implicate HIF1alpha as a 14q kidney cancer suppressor gene.Cancer Discov. 2011; 1: 222-235Google Scholar, 7Raval R.R. Lau K.W. Tran M.G. Sowter H.M. Mandriota S.J. Li J.L. Pugh C.W. Maxwell P.H. Harris A.L. Ratcliffe P.J. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma.Mol. Cell. Biol. 2005; 25: 5675-5686Google Scholar). This hypothesis is challenged by recent data generated in an animal model of ccRCC in which both isoforms appear to promote renal tumor development, assigning an even stronger oncogenic effect to HIF-1α than to HIF-2α (8Hoefflin R. Harlander S. Schafer S. Metzger P. Kuo F. Schonenberger D. Adlesic M. Peighambari A. Seidel P. Chen C.Y. Consenza-Contreras M. Jud A. Lahrmann B. Grabe N. Heide D. et al.HIF-1alpha and HIF-2alpha differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice.Nat. Scholar). The presence of of HIF which may in may In deletion of VHL in renal tubular cells leads to cell indicating that additional to promote tumor HIF activation A.P. Kaelin Jr., W.G. by the loss of the VHL tumor Scholar). In this of the HIF pathway by factors can in the HIF transcriptional and tumor Genetic important and several polymorphisms been to renal cancer development S. R. F. J.L. J. H. M. Ratcliffe P.J. D.R. J. renal cancer polymorphisms the HIF Scholar, S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar, J. C. Pugh C.W. Ratcliffe P.J. D.R. genetic at the renal cancer locus influence binding of HIF to an enhancer of Scholar, Li R. R. L. H. S. Ratcliffe P.J. D.R. of polymorphisms with HIF a pathway model of Scholar). analyses of several of suggest that a of in renal cancer may be by of the and of the HIF In this polymorphisms with HIF–DNA interactions at enhancers of important oncogenes as may influence of HIF genetic at the which for HIF-2α S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar, J. C. Pugh C.W. Ratcliffe P.J. D.R. genetic at the renal cancer locus influence binding of HIF to an enhancer of Scholar, G. M. J. M. Li P. et association identifies risk for renal cell Scholar, M. R. M. G. C.C. D. K. N. F. et a complex genetic for association with risk for renal cell Scholar, M. D. G. D. J. et association of renal cell carcinoma identifies on and 2011; Scholar). genetic an important for HIF in ccRCC development and may on events that renal tumor evolution from early lesions to of the of polymorphisms on HIF been in renal tubular cells that additional cell as factors may on the HIF pathway S. R. F. J.L. J. H. M. Ratcliffe P.J. D.R. J. renal cancer polymorphisms the HIF Scholar, S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar). In a recent of the genome-wide association in RCC patients has been for rs4903064, which is located in an of the DPF3 gene at G. M. J. M. Li P. et association identifies risk for renal cell Scholar). Double PHD is in and tissue, but and single cell DPF3 in the kidney and tubular cells, This protein is a member of the of SWI/SNF and has been to with of by PHD of transcription L. Li S. Zhou and regulation of binding by the PHD of Scholar). of DPF3 is observed in patients with but is the regulation of DPF3 in as the kidney renal cancer M. M. A. G. A. of a novel in 2013; Scholar). analyses from RCC suggest that the risk allele of is with DPF3 in renal but the is G. M. J. M. Li P. et association identifies risk for renal cell Scholar). we a functional of how DPF3 is to regulation by the HIF pathway in renal cells. We that the risk allele of a hypoxia-response element that HIF–DNA interactions at a enhancer of DPF3 The single nucleotide polymorphism (SNP) is located in the intron of the DPF3 gene indicating that genetic at this of DPF3 in renal cancer. for of DPF3 in cancer kidney tissue and in renal cancer we of DPF3 mRNA in a of clear and renal cancer and tissue specimens at the We of DPF3 in ccRCC to tissue of DPF3 was in RCC and in of DPF3 in ccRCC is in with data from the The Genome Atlas kidney renal clear cell carcinoma cohort to including and RCC In order to examine levels in DPF3 protein expression, we for DPF3 and DPF3 in a tumor renal the of mRNA we observed of DPF3 protein in the of tumor cells in ccRCC to the renal cancer and DPF3 is in we that this to the at G. M. J. M. Li P. et association identifies risk for renal cell Scholar). we from cohort for we observed an of ccRCC patients the risk allele to data from the cohort as a in ccRCC cohort to the levels of DPF3 mRNA in tumors from at 1 allele of of an gene by the In with we of the allele a for and DPF3 in the tumors A and the that it is the risk allele that is in we the of the in of DPF3 in from and tumor tissue from patients that for using a We a of DPF3 the in tumor from ccRCC patients to tissue tumor tissue from patients In with we DPF3 protein the in a of the for which we that of DPF3 is in tumors from ccRCC patients the risk allele of how the SNP with DPF3 expression, we genetic and data from for chromatin which we in primary renal tumor cells. We that are in with in the This in additional of the in a of chromatin in primary renal tumor cells by data We observed that of this is in the risk allele a recent of data from the this as a in ccRCC S. Zhou C. C.K. Cho et chromatin of primary 2018; Scholar). suggest that the SNP resides in a which as an enhancer of DPF3 of the rs4903064, we observed that the risk allele a novel element the creating a HIF-binding We HIF was in the effect of differentially chromatin at the we to and of from primary renal tumor and tubular cells isolated from for and allele-specific This an of chromatin the risk allele at in tumor cells and tubular cells with the to tubular cells and we a to the allele in chromatin in tubular cells with HIF data that to is HIF stabilization and that this effect is in ccRCC and tubular cells. for a of the we the enhancer with the of the and in cell and was by the under and the strong effect of HIF stabilization on allele-specific chromatin we the cells to the and in cells with the risk allele and the HIF-binding and that the effect was we of the in and cells, which for the HIF isoforms using This that the in was dependent on the presence of HIF-1α and Our data that HIF of DPF3 in ccRCC a element an by the risk allele of for an association of DPF3 protein levels with HIF in ccRCC we for DPF3, and HIF-2α in the We a of HIF-1α and DPF3 in ccRCC from the a function of HIF-1α on DPF3 with HIF-2α was but we HIF with the chromatin at the risk allele of We chromatin in primary renal tubular cells from with The interactions of HIF with this and binding of HIF to the risk allele a we binding of HIF to the factor enhancer and genotype-specific A was observed with HIF in isolated primary tumor cells by We HIF and chromatin immunoprecipitation by et and et from renal tumor cells and for at this J. K.J. J. Li Huang D. M. G. A.P. et enhancer activation of oncogenes in clear cell renal cell Discov. Scholar, M. N. R. Murray H. Ratcliffe P.J. D.R. of the and HIF-2alpha transcription factors in Scholar). In with we HIF interactions at this in the cell which is for the allele In in sequencing data from an isolated tumor we observed binding of both subunits to the enhancer we the of the of cells, we the risk allele in sequencing of the HIF interactions of HIF at the enhancer the cell K.W. R.R. Ratcliffe P.J. Pugh C.W. gene of hypoxia-inducible in renal cancer cells is by J. Cancer. Scholar, N. S. M. D. L. of a functional element that the of the J. 2005; Scholar). the that the HIF-binding at with a we observed levels of the active chromatin at this in tumors from ccRCC patients to tissue This effect was to that at the locus We sequencing data from et J. K.J. J. Li Huang D. M. G. A.P. et enhancer activation of oncogenes in clear cell renal cell Discov. Scholar). in renal cancer cells VHL to HIF levels 3, and This that in the with a VHL the of chromatin at the DPF3 HIF-binding that HIF interactions to enhancer for a for active in tumor cells from the the enhancer within the we observed for the which VHL of the that HIF-binding and enhancer in ccRCC cells, we allele-specific interactions of and in primary tubular cells for the This of the risk allele with HIF and as well as levels of at the risk allele HIF that HIF with the risk allele of and the enhancer and with the transcriptional examine functional of interactions on DPF3 expression, we DPF3 mRNA in primary tubular cells from large cohort of patients We determined that of cells to the HIF DPF3 in the risk allele to cells from for the allele We regulation of genes located the of DPF3 to this enhancer indicating that DPF3 is the stabilization of HIF to of DPF3 in that additional on DPF3 HIF the enhancer this effect is we chromatin at the enhancer and of DPF3 in a of cells. In chromatin at the DPF3 enhancer has in and cell to tubular cells of DPF3 HIF stabilization was in a of the cell with the from in which HIF stabilization regulate in cells with with the this that of DPF3 HIF stabilization in to be the of the on DPF3 mRNA levels in tumor cells to tubular cells We analyses on DPF3 to allele-specific in primary tubular and ccRCC cells. We confirmed of the risk allele in from primary tubular cells to to cells. we HIF-1α as the of DPF3 and of the isolated tumor cells HIF-1α to somatic we used mRNA as a for the of HIF-1α in cells. in cells with levels of to primary tubular cells of the we an in DPF3 the allele. we confirmed that allele-specific regulation of DPF3 by HIF-1α is to renal tubular cells and in ccRCC cells. how DPF3 on ccRCC development, we used of DPF3 in proximal renal tubular cells, from which ccRCC and We generated of cells for DPF3 from the cell and used of cells to cell with DPF3 than of the cells that in DPF3 in proximal tubular cells influence cell in of proximal tubular cell primary tubular cells from the of cells with DPF3 a to cells with In order to examine the effect of DPF3 on we for primary tubular cells We cells for DPF3 by using and a cells in the We used of the of and for at In with the from the growth we a in growth in cells with a DPF3 indicating that DPF3 is for growth of cells. we an SNP in the DPF3 gene with of HIF to the enhancer on the presence of the risk allele of rs4903064, which a and leads to of DPF3, which has in tubular cells. Our that the risk allele of an within an enhancer of the DPF3 HIF can to this and DPF3 mRNA in a genotype-specific increases in DPF3 mRNA are data additional for a in which genetic on the transcriptional of the HIF which in to development of ccRCC F. Ratcliffe P. I. The model of cancer Rev. Cancer. 2015; 15: Scholar). polymorphisms in with to renal cells S. R. F. J.L. J. H. M. Ratcliffe P.J. D.R. J. renal cancer polymorphisms the HIF Scholar, S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar). In with with an which has been identified as in a S. Zhou C. C.K. Cho et chromatin of primary 2018; Scholar). We observed in chromatin and in ccRCC cells and tubular cells. This effect to be dependent on the presence of the a functional and stabilization of this the hypothesis that HIF–DNA is for chromatin at this This is including that HIF to chromatin at which are already J. S. J. Pugh C.W. Ratcliffe P.J. D.R. genome-wide of HIF-binding by 2011; Scholar, A.L. binding of to active to Biol. Scholar). additional as transcription factors this effect at the DPF3 locus and to be The hypothesis of of factors be by the that the enhancer has in cells The that the effect of DPF3 regulation by HIF is in tubular cells that DPF3 dysregulation may to in the cancerous Our data in using proximal renal tubular cell and isolated tubular cells that DPF3 has the potential to growth of cells. This is in with a recent study, in which of DPF3 cell growth in the proximal tubular cell L. J. R. D.R. K. et regulation of DPF3, a member of the SWI/SNF the renal cancer J. Scholar). it to be DPF3 dysregulation is cancer to of tumor The that HIF-1α is to DPF3 from the RCC risk allele is in with an from the at which is by the HIF-1α which to the risk allele S. R. F. J.L. J. H. M. Ratcliffe P.J. D.R. J. renal cancer polymorphisms the HIF Scholar). HIF-1α operates at RCC risk is in to the hypothesis that HIF-1α as a tumor suppressor in ccRCC (6Shen C. Beroukhim R. Schumacher S.E. Zhou J. Chang M. Signoretti S. Kaelin Jr., W.G. Genetic and functional studies implicate HIF1alpha as a 14q kidney cancer suppressor gene.Cancer Discov. 2011; 1: 222-235Google Scholar). In this recent data from an animal model a for HIF-1α in tumor development (8Hoefflin R. Harlander S. Schafer S. Metzger P. Kuo F. Schonenberger D. Adlesic M. Peighambari A. Seidel P. Chen C.Y. Consenza-Contreras M. Jud A. Lahrmann B. Grabe N. Heide D. et al.HIF-1alpha and HIF-2alpha differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice.Nat. Scholar). In it is important to that deletions within both and DPF3 are frequent in ccRCC The of this for the of DPF3 and the of HIF-1α with DPF3 in of ccRCC is unclear but may to a of DPF3 in early of the We that HIF-1α is and that it in early of This hypothesis to be in of renal cancer We for of in we observed in cells in renal cells from with a HIF This effect was in cells with The that of DPF3 in a of cells in which the element is that the effect of on DPF3 mRNA is this element In with we regulation of the gene by the and HIF in the This that at on DPF3 HIF stabilization and that the enhancer the A recent on of polymorphisms has that to RCC development in Li P. M. L. D. A. B. G. et in association of renal cell J. Scholar). this effect in we the data from analyses in primary tubular cells to of the but in DPF3 levels and Our cohort be to in DPF3 this suggest that of HIF and the be in the The of DPF3 dysregulation on the and function of chromatin remodeling may influence renal cancer is that to mutations in SWI/SNF complex of DPF3 with function of the chromatin remodeling a of interactions SWI/SNF in cells that DPF3 with by in remodeling S. K. A. B. M. P. L. T. K. C. J. G. M. et characterization of mutations in Scholar). is to that of from the complex as mutations in PBRM1 to promote renal cancer In this of has been to of HIF genes in ccRCC the chromatin remodeling to the HIF pathway Li T. Kaelin Jr., W.G. of the PBRM1 tumor suppressor gene the in clear cell renal S. A. Scholar). we a in regulation of DPF3 mRNA we of isolated tumor cells to the protein indicating that DPF3 is dependent on in cells Our data on DPF3 gene regulation the element as an which with the potential of the HIF in an allele-specific in renal tubular cells. This a for the RCC risk by the and a for to the HIF and chromatin in the development of kidney tissue and tumor tissue from patients tumor was by the at the The at the of of the tissue, and and kidney and by an we used tumor tissue and kidney tissue from clear and renal cancer patients for tissue was used to using the and using renal tumors and renal tissue from the of the of of for tissue on and tumor been S. C. J. C. J. S. R. H. S. B. A. B. of regulation and the cell in renal cell 2015; Scholar). In to the of renal tumors and the by of tumor was to a in with the of for HIF-1α and HIF-2α on tissue as S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar). For DPF3, we used a DPF3 at a of and an by and on of and of cells using an to and for of an for in cancer Scholar). For of the HIF and DPF3 A.P. Kaelin Jr., W.G. by the loss of the VHL tumor Scholar, S. R. F. J.L. J. H. M. Ratcliffe P.J. D.R. J. renal cancer polymorphisms the HIF Scholar, S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar, J. C. Pugh C.W. Ratcliffe P.J. D.R. genetic at the renal cancer locus influence binding of HIF to an enhancer of levels of cell from and cells a from C. cells a from F. generated in the of M. and using with the large in in with and and levels of a for proximal renal cells. for and as S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar). For cells, we used and kidney tissue tumor tissue from patients tumor was used for primary cell as C. S. J. F. B. M. in and primary renal Scholar). and the of tissue was by the at the of with and and tubular cell in with and and 1 growth factor was confirmed by for and cell to 1 as to as using HIF-1α S. J.L. R. F. S. C. A. Ratcliffe P.J. D.R. J. Genetic at the renal cancer locus HIF binding to a Scholar, J. S. J. Pugh C.W. Ratcliffe P.J. D.R. genome-wide of HIF-binding by 2011; Scholar). used as as was as using in S. J. H. F. A. B. A. M.L. Ratcliffe P.J. D.R. J. hypoxia-inducible transcription factor of the Biol. Scholar). HIF-1α and HIF-2α subunits and HIF been C. J. M. the functional of hypoxia-inducible factor and HIF-2alpha by the of is a HIF-2alpha gene in and J. Scholar). at a of using from cells tissue was isolated using to the and using the transcription on a For to et with the following was for at using and was using of S. R. F. J.L. J. H. M. Ratcliffe P.J. D.R. J. renal cancer polymorphisms the HIF Scholar, P.G. of active from chromatin using of Scholar). We used a to for For allele-specific from and as well as was from from the was used in as an from cells for allele was used as using the The of allele for the from was to and the of from to the of from cells tumor tissue to the of the tissue For of cells, the of DPF3 to by the A of cells with using of cells generated by For mutation of was isolated by and the by was by in a to mutations S. J. H. F. A. B. A. M.L. Ratcliffe P.J. D.R. J. hypoxia-inducible transcription factor of the Biol. Scholar). of with mutations was by a and by sequencing of DPF3 was by of DPF3 in and cells was by the with a DPF3 A was determined using the and was for and for in at in tissue in was to the The of cells was by at using an and was by to and of the cell with DPF3 the used for the In well of a tissue a of and cell was and for at and cell and For cells and in cell and the was on on the to of cells to the of the for at of cell was was to from of the well using the and of using and to the from of an for the SNP and the using and using of and a in cells using with in was using a to in the cell of are in in and by the using a a HIF-2α We used a a and as analyses for using the the the with a of an the using using a renal cancer cells from under and at and cells to the as by et P.G. S. B. A. Cho M. et and of Scholar). using the and and using the and to the by using the and by et P.G. S. B. A. Cho M. et and of Scholar). for a of to and was by using to the and of the determined by using the of to from on a to a to the and using using with a and to The in by the a and by from the using We from and generated for using For sequencing in generated using to the of the using we with of and using the We used the following from renal cancer cells and in primary renal tubular data and to as from from data and data are This The We and for We the for and the at the for J. P. of this in of the for the This was by the for Research at the and the Research J. S. J. S. J. S. J. S. J. J. S. S. R. C. and A. K. data J. S. and S. G. R. K. C. S. M. M. A. and B. J. J. S. S. R. C. A. M. M. A. and B. and with and

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