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• W3083564694 abstract "Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel–Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1–actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo. Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC. Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel–Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1–actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo. Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC. The estimated incidence of and number of deaths from renal cell carcinoma (RCC) in the United States in 2019 are 73,280 and 14,770, respectively (1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2019.CA Cancer J. Clin. 2019; 69 (30620402): 7-3410.3322/caac.21551Crossref PubMed Scopus (10926) Google Scholar). The most common subtype, clear-cell RCC (ccRCC), occurs in >75% of RCC patients. Approximately 20–30% of those patients present with metastasis at the time of diagnosis. Another one-third of patients, following initial treatment, develop either local recurrence and/or distant metastases. Disquietingly, the 5-year survival of patients with advanced-stage ccRCC remains only 10% (2Jonasch E. Gao J. Rathmell W.K. Renal cell carcinoma.BMJ. 2014; 349 (25385470): g479710.1136/bmj.g4797Crossref PubMed Scopus (281) Google Scholar, 3Juengel E. Krueger G. Rutz J. Nelson K. Werner I. Relja B. Seliger B. Fisslthaler B. Fleming I. Tsaur I. Haferkamp A. Blaheta R.A. Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion.Oncotarget. 2016; 7 (26943029): 20410-2042410.18632/oncotarget.7804Crossref PubMed Scopus (5) Google Scholar, 4Pichler R. Heidegger I. Novel concepts of antiangiogenic therapies in metastatic renal cell cancer.Memo. 2017; 10 (29250198): 206-21210.1007/s12254-017-0344-2Crossref PubMed Scopus (9) Google Scholar). A distinguishing hallmark of ccRCC is its highly vascularized tumor microenvironment (TME) arising from the genetic loss of function (LOF) of the tumor-suppressor protein VHL (von Hippel–Lindau; inactivated in >90% of cases of sporadic ccRCC), leading to hypoxia-inducible factor-1 and -2 stabilization and up-regulation of the proangiogenic factor VEGF (4Pichler R. Heidegger I. Novel concepts of antiangiogenic therapies in metastatic renal cell cancer.Memo. 2017; 10 (29250198): 206-21210.1007/s12254-017-0344-2Crossref PubMed Scopus (9) Google Scholar). Although many ccRCC patients initially respond to therapies targeting VEGF or other proangiogenic signaling pathways, very few patients exhibit durable treatment-associated benefits, and virtually all develop progressive, drug-refractory disease (5Goel S. Duda D.G. Xu L. Munn L.L. Boucher Y. Fukumura D. Jain R.K. Normalization of the vasculature for treatment of cancer and other diseases.Physiol. Rev. 2011; 91 (21742796): 1071-112110.1152/physrev.00038.2010Crossref PubMed Scopus (982) Google Scholar, 6Bergers G. Hanahan D. Modes of resistance to anti-angiogenic therapy.Nat. Rev. 2008; 8 (18650835): 592-60310.1038/nrc2442Crossref Scopus (2231) Google Scholar, 7Welti J. Loges S. Dimmeler S. Carmeliet P. Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer.J. Clin. Invest. 2013; 123 (23908119): 3190-320010.1172/JCI70212Crossref PubMed Scopus (423) Google Scholar). Deeper molecular understanding of the pathogenesis and progression of ccRCC and its sequelae is expected to lead to the development of novel targeted interventions capable of providing improved clinical benefit. Dynamic control of the actin cytoskeleton is a key feature of all actin-dependent biological processes that includes cell migration and proliferation in both physiological and pathological contexts. Actin cytoskeletal regulation in cells involves the concerted actions of several major classes of actin-binding proteins (ABPs). Among them, the Pfn (profilin) family of ABPs play a key role in promoting actin polymerization in cells through their nucleotide-exchange activity on G-actin (facilitates ADP-to-ATP exchange on G-actin) and ability to act as a carrier of ATP–G-actin to a number of other actin assembly factors bearing polyproline motifs during F-actin elongation. Pfn's importance in the regulation of actin dynamics and actin-based cellular processes, such as cell migration and proliferation, is well-established in the literature (8Ding Z. Bae Y.H. Roy P. Molecular insights on context-specific role of profilin-1 in cell migration.Cell Adh. Migr. 2012; 6 (23076048): 442-44910.4161/cam.21832Crossref PubMed Scopus (54) Google Scholar, 9Ding Z. Gau D. Deasy B. Wells A. Roy P. Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells.Exp. Cell Res. 2009; 315 (19607826): 2963-297310.1016/j.yexcr.2009.07.004Crossref PubMed Scopus (43) Google Scholar, 10Ding Z. Lambrechts A. Parepally M. Roy P. Silencing profilin-1 inhibits endothelial cell proliferation, migration and cord morphogenesis.J. Cell Sci. 2006; 119 (16968742): 4127-413710.1242/jcs.03178Crossref PubMed Scopus (95) Google Scholar, 11Fan Y. Arif A. Gong Y. Jia J. Eswarappa S.M. Willard B. Horowitz A. Graham L.M. Penn M.S. Fox P.L. Stimulus-dependent phosphorylation of profilin-1 in angiogenesis.Nat. Cell Biol. 2012; 14 (23000962): 1046-105610.1038/ncb2580Crossref PubMed Scopus (48) Google Scholar, 12Kullmann J.A. Neumeyer A. Gurniak C.B. Friauf E. Witke W. Rust M.B. Profilin1 is required for glial cell adhesion and radial migration of cerebellar granule neurons.EMBO Rep. 2011; 13 (22081137): 75-8210.1038/embor.2011.211Crossref PubMed Scopus (29) Google Scholar, 13Böttcher R.T. Wiesner S. Braun A. Wimmer R. Berna A. Elad N. Medalia O. Pfeifer A. Aszódi A. Costell M. Fässler R. Profilin 1 is required for abscission during late cytokinesis of chondrocytes.EMBO J. 2009; 28 (19262563): 1157-116910.1038/emboj.2009.58Crossref PubMed Scopus (52) Google Scholar). Differential proteomic analyses of supernatants from the cultures of RCC cells versus normal kidney epithelial cells supported by RT-PCR and qualitative immunohistochemistry (IHC) analyses of a small number of RCC (14 cases) versus normal kidney tissues provided the first evidence of overexpression of Pfn1 (the major isoform of Pfn family of ABPs) in human ccRCC (14Minamida S. Iwamura M. Kodera Y. Kawashima Y. Ikeda M. Okusa H. Fujita T. Maeda T. Baba S. Profilin 1 overexpression in renal cell carcinoma.Int. J. Urol. 2011; 18 (21091798): 63-7110.1111/j.1442-2042.2010.02670.xCrossref PubMed Scopus (22) Google Scholar). Proteomic studies further identified Pfn1 to be one of the candidate markers of late stage ccRCC (15Masui O. White N.M. DeSouza L.V. Krakovska O. Matta A. Metias S. Khalil B. Romaschin A.D. Honey R.J. Stewart R. Pace K. Bjarnason G.A. Siu K.W. Yousef G.M. Quantitative proteomic analysis in metastatic renal cell carcinoma reveals a unique set of proteins with potential prognostic significance.Mol. Cell. Proteomics. 2013; 12: 132-14410.1074/mcp.M112.020701Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Neely B.A. Wilkins C.E. Marlow L.A. Malyarenko D. Kim Y. Ignatchenko A. Sasinowska H. Sasinowski M. Nyalwidhe J.O. Kislinger T. Copland J.A. Drake R.R. Proteotranscriptomic analysis reveals stage specific changes in the molecular landscape of clear-cell renal cell carcinoma.PLoS One. 2016; 11 (27128972): e015407410.1371/journal.pone.0154074Crossref PubMed Scopus (32) Google Scholar). In concordance with these findings, semiquantitative IHC studies performed on a larger cohort of patient samples (384 cases) subsequently established higher Pfn1 expression correlated with shorter overall survival (OS) and progression-free survival (PFS) of ccRCC patients (17Karamchandani J.R. Gabril M.Y. Ibrahim R. Scorilas A. Filter E. Finelli A. Lee J.Y. Ordon M. Pasic M. Romaschin A.D. Yousef G.M. Profilin-1 expression is associated with high grade and stage and decreased disease-free survival in renal cell carcinoma.Hum. Pathol. 2015; 46 (25704627): 673-680Crossref PubMed Scopus (17) Google Scholar). This particular study reported no statistically significant difference in % Pfn1low versus %Pfn1high expression between tumor and matched noncancerous regions of kidney in ccRCC patients. However, because those stratifications were based on percentages of Pfn1-immunoreactive regions on a semiquantitative evaluation scale, an accurate assessment of Pfn1 expression in different cell compartments in tumor versus normal adjacent regions of kidney in ccRCC patients is lacking in the literature. Overall, these studies suggest Pfn1's positive association with advanced disease features and adverse clinical outcomes in the setting of ccRCC. In this study, we first analyzed publicly available transcriptome data sets to identify other ABPs in addition to Pfn1 that have prognostic significance in ccRCC. We further demonstrate dysregulated expression of Pfn1 and a subset of those ABPs queried herein in tumor-associated (TA)–VECs in ccRCC patients. Additional experimental evidence developed in our studies support the role of Pfn1 in promoting proliferation and migration of RCC cells, and VEC-tumor cell cross-talk. Finally, we demonstrate proof-of-concept for a novel Pfn1–actin interaction inhibitor identified herein to reduce RCC cell aggressiveness in vitro and tumor growth in vivo. We first queried a The Cancer Genome Atlas (TCGA) transcriptome data set (downloaded from CBioportal) representing all 537 clinical cases of ccRCC to examine alteration characteristics (overexpression, gene amplification, down-regulation, deep-deletion, missense, and/or truncating mutations) of a total of eight major classes of actin cytoskeleton regulatory factors (Fig. 1). We included ABPs that are involved in (i) actin nucleotide exchange and shuttling (Pfn), (ii) G-actin sequestration (thymosin β4; maintains the cellular pool of polymerization-competent ATP-bound G-actin), (iii) actin nucleation (Arp2/3 complex, mammalian diaphanous (mDia)), (iv) activation of actin-nucleating factors (N-WASP (neural Wiskott–Aldrich syndrome protein), WAVE (WASP-associated verprolin homology)), (v) F-actin elongation (mDia and Ena (enabled)/VASP (vasodilator-stimulated phosphoprotein)), (vi) F-actin depolymerization and/or severing (cofilin and gelsolin), (vii) F-actin capping (CapZ), and (viii) F-actin cross-linking (filamin). As for Pfn, we restricted our analyses to Pfn1 and Pfn2 isoforms only because PFN3 transcript was undetectable, and the average transcript abundance of PFN4 (5.98 ± 5.45) was essentially negligible compared with that of either PFN1 (10958.0 ± 4594.6) or PFN2 (1821.1 ± 2996.3, expressed at a 6-fold lower abundance than Pfn1) in ccRCC tumors (Fig. 2A). Genes that are predominantly up-regulated (either through mRNA overexpression or gene amplification) include PFN isoforms (PFN1 and PFN2), ARPC3 (encodes Arp3 subunit of the Arp2/3 complex), DIAPH1 (encodes mDia1; exhibits the highest alteration frequency at 38%), DIAPH3 (encodes mDia3), ENAH (encodes Mena), VASP, EVL, CFL1 (encodes cofilin1), WASL (encodes N-WASP), and various CAPZ subunits. The most consistent down-regulated gene was FLNB (encodes Filamin-B), which involved deep deletion. For the remainder of the queried genes, no significant bias in any particular direction of change was evident. A subset of queried ABPs demonstrated association with disease features and/or clinical outcome in ccRCC (summarized in Table S1). Tumors of advanced Fuhrman grade (3 or 4) and stage (3 or 4) and associated with distant metastasis (M1) exhibited higher values of mRNA expression of PFN1 (but not the minor isoform PFN2), ARPC3, CFL1, CAPZ subunits (CAPZα1 and CAPZβ), and VASP relative to lower grade/stage (1 or 2) and nonmetastatic (M0) tumors, and these differences were statistically significant. Accordingly, higher expressions (more than the median value) of these genes also correlated with lower OS, as well as PFS of ccRCC patients. ENAH and EVL (the other two members of Ena/VASP protein family genes) also exhibited similar trends as seen for VASP with the only exceptions that ENAH expression was not different between M0 and M1 tumors and that high EVL expression only negatively impacted OS in a statistically significant manner (note however that EVL's association with PFS was very close to significance with p value equal to 0.056). Collectively, these data suggest that elevated expressions of PFN1, CFL1, CAPZ, ARPC3, and ENAH/VASP genes are poor prognostic features of ccRCC (disease association and survival plots pertinent to PFN1 are shown in Fig. 2 (B and C); disease association and survival plots for CFL1, CAPZ, ARPC3, and ENAH/VASP genes are shown in Figs. S1 and S2, respectively). We also found that expressions of a subset of ABP-encoding genes represent indicators of better prognosis in ccRCC (Table S1). Specifically, higher expressions of WASL (encodes N-WASP), WASF2 (encodes WAVE2), and GSN (encodes gelsolin) appeared to represent signatures of low-grade/stage and nonmetastatic tumors in association with improved survival (OS and/or PFS) in ccRCC patients. Furthermore, although the mDia1-encoding DIAPH1 gene is frequently overexpressed/amplified in ccRCC, higher expression of DIAPH1 also correlated with improved patient survival. Interestingly, there was no significant trend in DIAPH1 gene expression versus the clinicopathological features of tumors (survival curves for these four genes are shown in Fig. S3).Figure 2Adverse association of Pfn1 with patient outcome in human ccRCC. A, relative transcript abundance of different variants of Pfn in ccRCC. ***, p < 0.0001. B and C, association of transcript expressions of Pfn1 with disease features (grade, stage, metastasis) (B) and survival (OS and PFS) (C) of ccRCC patients (these data are based on the analyses of TCGA data representing 537 clinical cases). ***, p < 0.0001. Samples with missing stage, grade, and metastasis information were excluded (numbers in parentheses in B denote number of clinical cases in each category). For survival analyses (available for all 537 patients), transcript expression was dichotomized at the median value. Red and blue lines in Kaplan–Meier survival plots indicate higher and lower than median expression, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given that PFN1, ARPC3, ENAH/VASP, CFL1, and CAPZ represented poor prognostic indicators in the ccRCC setting, we further analyzed the TCGA transcriptome data set to examine the expression of these select ABP-encoding genes with clinical outcome in chromophobe (n = 63 patients) and papillary cell (n = 292 patients) histological subtypes of RCC. With the exception of ENAH, for which higher expression is correlated with shorter OS (p = 0.009) and PFS (p = 0.009), none of the other ABPs demonstrated any association with patient outcome in the setting of chromophobe RCC. However, we cannot dismiss the possibility that lack of clinical association of at least some of these ABP-encoding genes could be due to the small number of patient samples available for this subtype of RCC in the TCGA database. In the case of papillary cell RCC, higher expression of only ENAH and CAPZα1 genes correlated with statistically significant shorter OS (p values equal to 0.025 and 0.019 for ENAH and CAPZα1, respectively), whereas higher expression of only EVL and ARPC3 genes was associated with diminished PFS (p values equal to 0.04 and 0.05, respectively). Therefore, adverse clinical outcome associated with elevated tumor expression of PFN1, VASP, and CFL1 genes appears to be restricted solely to the clear-cell histological subtype. We focused subsequent studies on Pfn1 because of concordance of our transcriptome-based findings with previous proteomic and semiquantitative IHC-based correlation of Pfn1 expression with advanced disease features and adverse patient outcome from other independent studies (15Masui O. White N.M. DeSouza L.V. Krakovska O. Matta A. Metias S. Khalil B. Romaschin A.D. Honey R.J. Stewart R. Pace K. Bjarnason G.A. Siu K.W. Yousef G.M. Quantitative proteomic analysis in metastatic renal cell carcinoma reveals a unique set of proteins with potential prognostic significance.Mol. Cell. Proteomics. 2013; 12: 132-14410.1074/mcp.M112.020701Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Neely B.A. Wilkins C.E. Marlow L.A. Malyarenko D. Kim Y. Ignatchenko A. Sasinowska H. Sasinowski M. Nyalwidhe J.O. Kislinger T. Copland J.A. Drake R.R. Proteotranscriptomic analysis reveals stage specific changes in the molecular landscape of clear-cell renal cell carcinoma.PLoS One. 2016; 11 (27128972): e015407410.1371/journal.pone.0154074Crossref PubMed Scopus (32) Google Scholar, 17Karamchandani J.R. Gabril M.Y. Ibrahim R. Scorilas A. Filter E. Finelli A. Lee J.Y. Ordon M. Pasic M. Romaschin A.D. Yousef G.M. Profilin-1 expression is associated with high grade and stage and decreased disease-free survival in renal cell carcinoma.Hum. Pathol. 2015; 46 (25704627): 673-680Crossref PubMed Scopus (17) Google Scholar). To determine which cells in the ccRCC TME preferentially overexpressed Pfn1, we next performed a qualitative IHC assessment of tissue microarrays (TMAs) containing a large cohort (n = 417) of ccRCC tumors (Fig. 3A), using a Pfn1 antibody that we used for IHC in our previous studies (18Gau D.M. Lesnock J.L. Hood B.L. Bhargava R. Sun M. Darcy K. Luthra S. Chandran U. Conrads T.P. Edwards R.P. Kelley J.L. Krivak T.C. Roy P. BRCA1 deficiency in ovarian cancer is associated with alteration in expression of several key regulators of cell motility: a proteomics study.Cell Cycle. 2015; 14 (25927284): 1884-189210.1080/15384101.2015.1036203Crossref PubMed Scopus (20) Google Scholar, 19Chakraborty S. Jiang C. Gau D. Oddo M. Ding Z. Vollmer L. Joy M. Schiemann W. Stolz D.B. Vogt A. Ghosh S. Roy P. Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells.Br. J. Cancer. 2018; 119 (30318519): 1106-111710.1038/s41416-018-0284-6Crossref PubMed Scopus (8) Google Scholar). By immunoblot analyses of cell lysates in Pfn-isoform–specific gene knockdown settings, we also previously confirmed that this antibody does not recognize Pfn2 (20Joy M. Gau D. Castellucci N. Prywes R. Roy P. The myocardin-related transcription factor MKL co-regulates the cellular levels of two profilin isoforms.J. Biol. Chem. 2017; 292 (28546428): 11777-1179110.1074/jbc.M117.781104Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Furthermore, human tumor sections (kidney or ovarian) incubated with secondary antibody alone (i.e. without the presence of Pfn1 antibody) did not result in either chromagen- or fluorescence-based IHC signal, further establishing the specificity of our IHC staining (Fig. S4). Pfn1 IHC of ccRCC TMA revealed three distinct patterns of Pfn1 expression: (i) negligible Pfn1 expression throughout the tumor (5.8% frequency), (ii) strong Pfn1 expression in stromal cells (VEC, lymphocytes) but negligible expression in tumor cells (64.5% frequency), and (iii) strong Pfn1 expression in both stromal and tumor cells (29.7% frequency). In the latter sample cohort, 71% of specimens exhibited a nuclear distribution of Pfn1 expression in tumor cells, with the remaining 29% of specimens characterized by nucleocytoplasmic staining of tumor cells. These findings suggest that the vast majority of ccRCC tumors display a pattern of strong Pfn1 expression selectively in stromal cells within the TME; this is in accord with the general assessment of Minamida et al. (14Minamida S. Iwamura M. Kodera Y. Kawashima Y. Ikeda M. Okusa H. Fujita T. Maeda T. Baba S. Profilin 1 overexpression in renal cell carcinoma.Int. J. Urol. 2011; 18 (21091798): 63-7110.1111/j.1442-2042.2010.02670.xCrossref PubMed Scopus (22) Google Scholar) in their qualitative evaluation of a limited number of ccRCC samples. Notably, patients with tumors graded for coordinate Pfn1 positivity in all three cellular compartments (VECs, lymphocytes, and tumor cells) tended to have reduced OS when compared with patients with tumors in which only stromal cells were graded as Pfn1-positive (p = 0.168; Fig. 3B). Although difference in the survival characteristics between the two cellular patterns of Pfn1 expressions was not statistically significant, we speculate that may be due to limitations in qualitative characterization of Pfn1 expression in our IHC studies of tumor specimens. Given the prevalence of a stromal cell–associated signature for Pfn1 overexpression in ccRCC, we next performed gene expression profiling of flow-sorted CD34+CD45negCD146+ VEC isolated from freshly isolated enzymatically digested, ccRCC tumors, which revealed a robust (5.3–21.7-fold) up-regulation in Pfn1 expression in tumor-associated TA-VEC relative to VEC sorted from patient-matched normal adjacent tissue (NAT-VEC; Fig. 3C). Among the other poor prognostic cytoskeletal markers, only Mena, CapZα1, and CapZβ were also determined to be strongly up-regulated in tumor-associated VEC (TA-VEC) versus normal adjacent tissue VEC (NAT-VEC) (Fig. 3C). In contrast, a parallel analysis of flow-sorted CD34negCD45negCD146+ vascular pericytes isolated from tumor versus NAT samples showed no significant differences in the expression of these proteins (data not shown). Because Pfn1 interacts with Mena/VASP proteins and is a major promoter of Mena/VASP-mediated actin assembly and actin-driven cellular processes (21Gau D. Veon W. Shroff S.G. Roy P. The VASP-profilin1 (Pfn1) interaction is critical for efficient cell migration and is regulated by cell-substrate adhesion in a PKA-dependent manner.J. Biol. Chem. 2019; 294 (30814249): 6972-698510.1074/jbc.RA118.005255Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), transcriptional dysregulation of the Pfn1:Mena/VASP cytoskeletal pathway in TA-VEC could represent a bio-signature of poor prognosis in ccRCC. A previous study correlated elevated serum levels of Pfn1 (sPfn1) with the degree of atherosclerosis in humans (22Caglayan E. Romeo G.R. Kappert K. Odenthal M. Südkamp M. Body S.C. Shernan S.K. Hackbusch D. Vantler M. Kazlauskas A. Rosenkranz S. Profilin-1 is expressed in human atherosclerotic plaques and induces atherogenic effects on vascular smooth muscle cells.PLoS One. 2010; 5 (21049052): e1360810.1371/journal.pone.0013608Crossref PubMed Scopus (41) Google Scholar), suggesting that sPfn1 has potential to serve as a cytoskeletal biomarker in certain disease contexts. We performed exploratory ELISA to compare sPfn1 levels between small cohorts of stage 4/M1 ccRCC patients (n = 29; 8 female, 21 male; median age, 59 years; range, 45–67 years) and age-matched normal human donors (n = 11; 5 male, 6 female; median age, 60 years; range, 48–76 years) as control. The ccRCC patients had completed treatment with high-dose interleukin-2 combined with the autophagy inhibitor hydroxychloroquine. Pfn1 was detectable in both normal donors and ccRCC patients in the ng/ml range. Despite the small sample sizes evaluated, the mean sPfn1 level in ccRCC patients at baseline/pretreatment (29.6 ± 15.3 ng/ml) was ∼2-fold higher (p = 0.0003) versus normal donors (15.83 ± 7.62 ng/ml; Fig. S5). Although the significant overlap of sPfn1 level between normal and patient cohorts precludes us from any definitive conclusion at this point, our preliminary indication of a trend of elevated sPfn1 level in ccRCC patients justifies future studies involving a larger cohort of subjects to examine the potential utility of sPfn1 as a predictive biomarker in ccRCC. Next, to determine whether Pfn1 has any role in proliferation of ccRCC cells, we first studied the effect of transient silencing Pfn1 expression on proliferation of RVN (a variant of the widely used murine RENCA RCC cell line engineered for VHL gene deletion by CRISPR/Cas9) and 786-0 (a VHL-negative human cell line originally derived from ccRCC clinical specimen) cell lines, respectively, in 2D culture. Note that although the parent RENCA (VHLWT) cell line has been considered poorly reflective of human ccRCC, RVN (VHL−/−) cells overexpress many of the genes associated with aggressive ccRCC in humans and appear to represent an appropriate surrogate of human disease (23Hu J. Schokrpur S. Archang M. Hermann K. Sharrow A.C. Khanna P. Novak J. Signoretti S. Bhatt R.S. Knudsen B.S. Xu H. Wu L. A non-integrating lentiviral approach overcomes Cas9-induced immune rejection to establish an immunocompetent metastatic renal cancer model.Mol. Ther. Methods Clin. Dev. 2018; 9 (29766028): 203-21010.1016/j.omtm.2018.02.009Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 24Schokrpur S. Hu J. Moughon D.L. Liu P. Lin L.C. Hermann K. Mangul S. Guan W. Pellegrini M. Xu H. Wu L. CRISPR-mediated VHL knockout generates an improved model for metastatic renal cell carcinoma.Sci. Rep. 2016; 6 (27358011): 2903210.1038/srep29032Crossref PubMed Scopus (25) Google Scholar). Proliferation of both VHL−/− cell lines was reduced when Pfn1 expression was suppressed (Fig. 4, A and B). Pfn1's interactions with actin and other actin assembly factors (such as Mena/VASP proteins) play important roles in actin polymerization at the leading edge and membrane protrusion (a key step of cell migration) (21Gau D. Veon W. Shroff S.G. Roy P. The VASP-profilin1 (Pfn1) interaction is critical for efficient cell migration and is regulated by cell-substrate adhesion in a PKA-dependent manner.J. Biol. Chem. 2019; 294 (30814249): 6972-698510.1074/jbc.RA118.005255Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 25Rotty J.D. Wu C. Haynes E.M. Suarez C. Winkelman J.D. Johnson H.E. Haugh J.M. Kovar D.R. Bear J.E. Profilin-1 serves as a gatekeeper for actin assembly by Arp2/3-dependent and -independent pathways.Dev. Cell. 2015; 32 (25543281): 54-6710.1016/j.devcel.2014.10.026Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). In most physiological contexts, LOF of Pfn1 leads to defects in membrane protrusion and cell migration (8Ding Z. Bae Y.H. Roy P. Molecular insights on context-specific role of profilin-1 in cell migration.Cell Adh. Migr. 2012; 6 (23076048): 442-44910.4161/cam.21832Crossref PubMed Scopus (54) Google Scholar, 9Ding Z. Gau D. Deasy B. Wells A. Roy P. Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells.Exp. Cell Res. 2009; 315 (19607826): 2963-297310.1016/j.yexcr.2009.07.004Crossref PubMed Scopus (43) Google Scholar, 10Ding Z. La" @default.
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• W3083564694 title "Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells" @default.
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• W3083564694 doi "https://doi.org/10.1074/jbc.ra120.013963" @default.
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