Abstract B52: Huntingtin interacting protein 1 (HIP1) mediates prostate carcinogenesis and progression through effects on signal transducers and activators of transcription (STATs)
Authors: Rao Roheet Bantval , Wang Jianghua , Berry Lorraine S. , Reichelt Stefanie , Ittmann Michael M. , Neal David E. , Mills Ian G. 
Published: June 2012

Background: HIP1 is an adaptor protein classically involved in clathrin mediated endocytosis by acting as a scaffolding factor. Its overexpression has been linked to a multitude of epithelial cancers including prostate cancer (CaP)(1). FGFR4 stabilisation by HIP1 in non-transformed PNT1 a cells was implicated in its role in CaP(2). The molecular mechanisms that mediate the effects of HIP1 overexpression however, remain largely unknown.

Methods: PNT1a- a non-transformed prostate epithelial cell line stably overexpressing HIP1 (PNT1a-HIP1) or empty vector (control) was used in anchorage independent growth, and migration assays to investigate functional effects; IIIumina bead arrays to determine gene expression; Human Phospho-Kinase Array(R&D systems) to detect site-specific phosphorylation of 46 protein kinase sites; iCys imaging for quantification of IF to determine nuclear STAT protein; rtPCR and western blots for mRNA and protein respectively.

Results: Analysis of downstream effects of FGFR4 stabilisation by HIP1 using the Human Phospho-Kinase Array revealed significant differences in STAT phosphorylation. Knocking down (KD) HIP1 in PNT1a-HIP1 cells reversed these phosphorylation events. Since STATs undergo nuclear translocation to cause transcription we assessed nuclear STAT levels and observed significantly increased nuclear STATs in PNT1a-HIP1. We also observed increased nuclear STAT in cells treated with FGF2 following serum starvation indicating FGFR mediated STAT phosphorylation. To test whether JAKs acting downstream of FGFR mediated STAT phosphorylation, we used a JAK-STAT kinase inhibitor and observed reduction in phospho-STATs and nuclear STAT proteins. Cellular transformation of PNT1 a-HIP1-the ability to form colonies in soft agar, was only partially abrogated by FGFR4 KD (2) in previous studies. However, inhibitors of STAT phosphorylation completely abrogated soft agar colony formation, while also significantly reducing cell migration of the PNT1a-HIP1. Indeed, PNT1a-HIP1 were significantly more sensitive (EVIC50 &gt; HIP1 IC50) to STAT inhibition. We tested this in DU145, a highly transformed, invasive androgen-receptor negative CaP cell line, which has constitutively high levels of STAT phosphorylation. Interestingly, HIP1 knockdown in DU145 caused a reduction in STAT phosphorylation, soft agar colony formation, and cell migration. Using unbiased gene expression arrays and stringent cut-offs (p &lt;0.01; −0.05&lt;log FC&gt;0.6) to generate a list of differentially expressed genes, we observed a significant upregulation of TGFβ superfamily ligands which have been implicated to have paracrine effects on CaP growth and progression(3). To test these paracrine effects on classical androgen receptor positive cell lines, we cultured LNCaPs with conditioned media from PNT1 a-HIP1 and observed a significant increase in growth. Taken together, phospho-STATs affect TGFβ signalling which contributes to CaP progression.

Conclusion: We believe HIP1 overexpression mediates its effects through constitutive activation of STATs, which contributes to cancer progression, and drug resistance through activation of autocrine and paracrine TGFβ signalling molecules. Future work will assess HIP1 as a predictor of chemoresistance and a potential role for STAT inhibitors as an adjuvant for treatment of aggressive drug resistant CaP.

1. D. S. Rao et al., J Clin Invest 110, 351 (Aug, 2002).

2. J. Wang, W. Yu, Y. Cai, C. Ren, M. M. Ittmann, Neoplasia 10, 847 (Aug, 2008).

3. A. R. Bauskin et al., Cancer Res 66, 4983 (May 15, 2006).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B52.