1. page 4 line 16 RCC has highest incidence in the development.. development

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1 Author s response to reviews Title: Insulin-like growth factor-1 signaling in renal cell carcinoma Authors: Adam Tracz (a.f.tracz@gmail.com) Cezary Szczylik (cszczylik@wim.mil.pl) Camillo Porta (c.porta@smatteo.pv.it) Anna Czarnecka (anna.czarnecka@gmail.com) Version: 1 Date: 01 Apr 2016 Author s response to reviews: Dear Prof. Solera, Reviewer #1: The authors have discussed the role of IGF-1 signaling in renal cell carcinoma. The manuscript is well written and discusses with appropriate literature the relevance of the pathway and its implication in carcinoma. There are however a few minor corrections in the MS 1. page 4 line 16 RCC has highest incidence in the development.. development Has been corrected to RCC has highest incidence in highly developed countries 2. page 4 line 25 have a significant effect on menaagment of RCC. Menagement Has been corrected to have a significant effect on management of RCC 3. Briefly discuss table-1 in the text. The list of drugs used for renal cell cancer treatment (presented in Table 1) has been described in the text: There are 3 major groups of systematic treatment that are used for metastatic RCC:

2 cytokines, mtor inhibitors and anti- vascular endothelial growth factor (VEGF) - targeted drugs [2]. Cytokine based immunotherapies included interferon-alpha (1) and high dose interleukin 2 (IL-2) (2), while mtor inhibitors approved for RCC treatment are everolimus (8) and temsirolimus (9). VEGF pathway inhibitors used are 1) tyrosine kinase inhibitors as sorafenib (3), sunitinib (4), pazopanib (5), and axitinib (6), and 2) anti-vegf monoclonal antibody bevacizumab (7) (Table 1). 4. Discuss briefly the molecular structure of IGF-1 and insulin and their homology in the Section "Great homology - IGF-1 receptor and insulin receptor" The homology has been described and this paragraph has been re-written and supplemented with requested data IGFR-1 is a transmembrane receptor with tyrosine kinase activity and is built of two α-subunits (located extracellularly) and two β-subunits (spanning the membrane and activating intracellular signal transduction). Both the α and β subunits are synthesized from a single precursor mrna. IGF1R shares a high structural homology with the insulin receptor (IR) has more than 50% in the overall amino acid sequence and in particular 84% similarity in the tyrosine kinase domain and 45 65% in the ligand-binding domain. Moreover ligand-dependent activation of the IGF1R and IR activates almost identical downstream signaling pathways (27). After IGF-1 binging activation of tyrosine kinase (β-subunits) results in downstream signaling via IR substrate proteins (IRS1-4), Src homology 2 domain containing transforming protein 1 (Shc), GRB2-associated binding protein 1 (Gab-1), Casitas B-lineage Lymphoma protooncogene E3 ubiquitin protein ligase (Cbl), Phosphatidyl Inositol 3-Kinase (PIK3), Protein kinase B (Akt), mammalian target of rapamycin (mtor), mitogen-activated protein kinase (MAPK) and signal regulatory protein family (28). Insulin and IGFs have a great homology and can have cross-reactivity upon receptors. Moreover hybrid receptors - constituted of IR and IGF1R heterodimers have been shown to have cellular biological effects resembling those of the IGF1R and were found in colon cancer, thyroid cancer and breast cancer cell lines and tissues (29). To complicate the interaction even more there are two IR isoforms, arising in the cell by alternative splicing of exon 11 isoform IR-A, that lacks exon 11, and isoform IR-B containing exon 11. Insulin does not bind to the hybrid receptors, but binds to IR-A, IR-B, and IGF-1R but binds to the IGF-1R with much lower affinity than to the IR. IGF-I binds to the IGF- 1R, hybrid receptors, and IR but has much lower affinity for the IR than IGF-1R (30). In total insulin and IGF-1 interact with six receptors: the type I IGF receptor (IGF1R), the IRA (IR-A, predominantly expressed in fetal tissue), the IRB (IR-B, predominantly expressed in adult tissue), hybrid receptors of IGF and IR-A, hybrid receptors of IGF and IR-B, and hybrid receptors of IR-A and IR-B (31, 32). Insulin and IGF-1 while binding to IGF1R, IR-A, IGF1R/IR-A, mediate mostly mitogenic signaling (Ras>MEK>Erk1/2 pathway), while binding to IR-B activate mostly metabolic pathway (PI3K>Akt>mTOR) (17, 28, 33). As a result both insulin and IGF-1 can act through the hybrid receptors and through the specific receptor for their ligand.

3 5. Page-6 line 23 Deregulated IGF1R kinas. Has been corrected to Deregulated IGF1R kinase activity and its overexpression is linked with many cancers 6. Page-6 line 29 promoter sequence is lack of TATA.. lacks Has been corrected to Unlike in most genes, promoter sequence lacks TATA and CCAAT boxes 7. A section about epigenetic regulation can be presented. Epigenetic regulation of IGF pathway in RCC has been presented including: Werner H, Sarfstein R: Transcriptional and epigenetic control of IGF1R gene expression: implications in metabolism and cancer. Growth Horm IGF Res 2014, 24(4): Perks CM, Holly JM: Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer. Journal of cell communication and signaling 2015, 9(2): Banks RE, Tirukonda P, Taylor C, Hornigold N, Astuti D, Cohen D, Maher ER, Stanley AJ, Harnden P, Joyce A et al: Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer. Cancer Res 2006, 66(4): Reviewer #2: This article colligates correlated documents and write the review.it showed that IGF-1 plays an important role in protection from apoptosis and regulation of cell growth, then the relavant signaling was reviewed.that is a good work,however, some revision are also needed to polish the story. 1.In this article, it shows the role of IGF-1 signaling in renal cell carcinoma, I think it is necessary to show us the structure of IGF-1 for describing the general IGF-1 function

4 IGF-1 section has been developed Insulin-like growth factor 1 (IGF-1, somatomedin C) is a natural anabolic peptide hormone produced mainly by hepatocytes. IGF-1 with molecular weight of 7649 Da is built by 70 amino acids and single polypeptide chain with three intramolecular disulfide bridges. Production of IGF-1 is stimulated by growth hormone (GH) secreted by anterior pituitary. IGF-1 production is also stimulated by insulin and has influenced on reduction of lipolysis, glycolysis, inhibition of lipolytic function of adrenaline, embryonic growth and differentiation of cells. IGF-1 may also be released independently of GH. Circulating IGF-1 produced in liver acts in endocrine manner, but locally produced IGF-1 acts also in an autocrine manner. IGF functions therefore both as circulating hormone and tissue growth factor. Circulating IGF-1 forms a complex with two other proteins the IGF binding protein (IGFBP) and the acid labile subunit (ALS). Six different IGFBPs were characterized, but about 75% of serum IGFs are bound to IGFBP3 and only 1% of serum IGF-1 is free-bioactive form [24]. IGFBPs are also mostly synthesized in the liver. Nevertheless IGFs and IGFBPs are also produced in other organs, acting locally in autocrine and paracrine manner and mediating stromal - epithelial cell interactions [25]. IGFBPs acts in a competing manner against IGFR (IGF receptors) and IGFBP proteases. IGF-1 and IGFBP-3 complex play crucial role in mitogenesis, cell differentiation and survival [26]. IGF-1 null mice die shortly after birth [27]. 2.I find that IGF-1 could induce osteogenic differentiation in mesenchymal stem cells through enhancing BMPs/Smads signaling from the recently published article in the journal "BMB Rep", I think that this signal may be an important pathway for tumor progression, however, I don`t find revelvant description in the article. Sarcomatoid renal cell carcinoma is an uncommon renal tumor, and osteogenic differentiation has been reported in only a few of these tumors. Culturing normal renal and ccrcc cells in differentiation media showed that only ccrcc cells were induced to undergo osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified including significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2). Pathway signature and cellular differentiation in clear cell renal cell carcinoma. Tun HW, Marlow LA, von Roemeling CA, Cooper SJ, Kreinest P, Wu K, Luxon BA, Sinha M, Anastasiadis PZ, Copland JA. PLoS One May 18;5(5):e I think it is necessary to add the Figure legend for the figures.

5 Figure legends have been prepared: Figure 1. Schematic representation of downstream signaling of IGF1R. AKT, protein kinase B; AMPK, AMP-activated protein kinase; Bcl-2, B-cell lymphoma 2; BAD, B-cell CLL/lymphoma 2 antagonist of cell death; ERK 1/2, extracellular-signal-regulated kinase 1/2, IGF1R, insulinlike growth factor 1 receptor; IR, insulin receptor; IRS1-4, insulin-like receptor substrate 1-4; MEK, mitogen-activated protein kinase kinase; mtor, mammalian target of rapamycin; PI3K/AKT, phosphatidylinositol 3-kinase/AKT; PDK1, 3-phosphoinositide-dependent protein kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5- trisphosphate; PTEN, phosphatase and tensin homolog; GLUT4, Glucose transporter type 4; HIF-1α, Hypoxia-inducible factor 1-alpha, VEGF, Vascular endothelial growth factor; Figure 2. Schematic representation of promoter region of IGF1R and its main transcription factors. Sp1, Specificity protein 1; HMGA1, High mobility group A1; KLF6, Krüppel-like factor 6; E2F1, E2F family of transcription factors; POL, RNA polymerase II; TBP, TATA-binding protein; GC, GC boxes; INR, initiator element. Figure 3. Regulation of promoter activity of IGF1R gene by tumor suppressor genes. POL, RNA polymerase II; TBP, TATA-binding protein; GC, GC boxes; INR, initiator element. 4.In this article, the references are too old, it should cite several new published studies. More current research has been cited including: Brahmkhatri VP, Prasanna C, Atreya HS: Insulin-like growth factor system in cancer: novel targeted therapies. BioMed research international 2015, 2015: Vigneri PG, Tirro E, Pennisi MS, Massimino M, Stella S, Romano C, Manzella L: The Insulin/IGF System in Colorectal Cancer Development and Resistance to Therapy. Frontiers in oncology 2015, 5:230. Perks CM, Holly JM: Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer. Journal of cell communication and signaling 2015, 9(2): Weinstein D, Sarfstein R, Laron Z, Werner H: Insulin receptor compensates for IGF1R inhibition and directly induces mitogenic activity in prostate cancer cells. Endocrine connections 2014, 3(1):24-35

6 Werner H, Sarfstein R: Transcriptional and epigenetic control of IGF1R gene expression: implications in metabolism and cancer. Growth Horm IGF Res 2014, 24(4): Kamenicky P, Mazziotti G, Lombes M, Giustina A, Chanson P: Growth hormone, insulin-like growth factor-1, and the kidney: pathophysiological and clinical implications. Endocr Rev 2014, 35(2): Lkhagvadorj S, Oh SS, Lee MR, Jung JH, Chung HC, Cha SK, Eom M: Insulin receptor expression in clear cell renal cell carcinoma and its relation to prognosis. Yonsei Med J 2014, 55(4): Gristina V, Cupri MG, Torchio M, Mezzogori C, Cacciabue L, Danova M: Diabetes and cancer: A critical appraisal of the pathogenetic and therapeutic links. Biomed Rep 2014, 3(2): A. Czarnecka