Oncogene (2012) 31, 4472–4483 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE DNAJB6 chaperones PP2A mediated dephosphorylation of GSK3b to downregulate b-catenin transcription target, osteopontin A Mitra1, ME Menezes1, LK Pannell1, MS Mulekar2, RE Honkanen3, LA Shevde1 and RS Samant1 1Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA; 2Department of Mathematics and Statistics, University of South Alabama, Mobile, AL, USA and 3Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA Elevated levels of the oncoprotein, osteopontin (OPN), Introduction are associated with poor outcome of several types of cancers including melanoma. We have previously reported The human genome codes for more than 40 distinct an important involvement of DNAJB6, a member of heat- heat-shock protein 40 (HSP40) (DNAJ) members (Mitra shock protein 40 (HSP40) family, in negatively impacting et al., 2009). A typical DNAJ protein is characterized by tumor growth. The current study was prompted by our a J domain, a glycine-Phenylalanine (G/F) domain and observations reported here which revealed a reciprocal a C-terminal domain. Depending on the presence of relationship between DNAJB6 and OPN in melanoma these domains, this family is divided into Type I, Type II specimens. The ‘J domain’ is the most conserved domain and Type III (also known as DNAJ A, B and C) types of HSP40 family of proteins. Hence, we assessed the (Ohtsuka and Hata, 2000). The J domain consists of functional role of the J domain in activities of DNAJB6. highly conserved Histidine-Proline-Aspartic acid (HPD) We report that the J domain of DNAJB6 is involved in tri-peptide and, is the most conserved domain of this mediating OPN suppression. Deletion of the J domain family and has been implicated in providing the renders DNAJB6 incapable of impeding malignancy and substrate specificity to the individual members (Kelley, suppressing OPN. Our mechanistic investigations reveal 1998). The importance of this family in critical that DNAJB6 binds HSPA8 (heat-shock cognate protein, biological activities has been highlighted in the recent HSC70) and causes dephosphorylation of glycogen past (Edwards and Munger, 2004; Tsai et al., 2006; synthase kinase 3b (GSK3b) at Ser 9 by recruiting protein Dhennin-Duthille et al., 2011). Noteworthy among them phosphatase, PP2A. This dephosphorylation activates are the recruitment of HSP70, participation in guiding GSK3b, leading to degradation of b-catenin and subse- misfolded proteins to degradation, cell-cycle control and quent loss of TCF/LEF (T cell factor1/lymphoid enhancer regulation of protein kinases (Kelley, 1998; Vos et al., factor1) activity. Deletion of the J domain abrogates 2008; Kampinga and Craig, 2010). DNAJB6, the focus assembly of this multiprotein complex and renders GSK3b of this study, has been reported to reduce protein inactive, thus, stabilizing b-catenin, a transcription co- aggregates in Huntington’s, Parkinson’s diseases, pla- activator for OPN expression. Our in-vitro and in-vivo cental development and neural stem cells (Hunter et al., functional analyses show that silencing OPN expression in 1999; Chuang et al., 2002; Dai et al., 2005; Watson et al., the background of deletion of the J domain renders the 2007, 2009; Zhang et al., 2008; Dey et al., 2009). We resultant tumor cells less malignant despite the presence of have previously demonstrated the role of DNAJB6 in stabilized b-catenin. Thus, we have uncovered a new reducing tumorigenicity and metastatic potential (Mitra mechanism for regulation of GSK3b activity leading to et al., 2008). Our studies have also shown that DNAJB6 inhibition of Wnt/b-catenin signaling. can cause reversal of epithelial-mesenchymal transition Oncogene (2012) 31, 4472–4483; doi:10.1038/onc.2011.623; (EMT) by inhibiting Wnt/b-catenin signaling (Mitra published online 23 January 2012 et al., 2010). Melanoma is a highly invasive and metastatic type of Keywords: DNAJB6; cancer; SPP1; OPN; GSK3b; skin cancer. Cure rates drop precipitously once it has PP2A metastasized. Many distinct signaling mechanisms involving molecules such as b-Raf, AKT, P16 and P53 have been implicated to be involved in melanoma progression (Rodolfo et al., 2004; Lopez-Bergami et al., 2008; Martin et al., 2009). Osteopontin (OPN), is an RGD domain-containing SIBLING family glyco- Correspondence: Professor RS Samant, Department of Oncologic protein that has diverse roles in promoting malignancy Sciences, Mitchell Cancer Institute, University of South Alabama, of a multiple tumor types and is advocated to be 1660 Spring Hill Avenue, Mobile, AL 36604, USA. E-mail: [email protected] involved in melanoma progression and metastasis Received 5 July 2011; revised 28 November 2011; accepted 29 November (Buback et al., 2009; Shevde et al., 2010). We have 2011; published online 23 January 2012 previously shown that OPN expression is increased with DNAJB6 multiprotein complex with GSK3b regulates OPN A Mitra et al 4473 progression and gain of metastatic potential by melano- assessed DNAJB6 and OPN expression from melanoma mas (Riker et al., 2008; Metge et al., 2010). OPN has been specimens and found that OPN expression was sig- implicated to be involved in promoting immune evasion nificantly upregulated in stage III and IV metastatic by tumor cells, in EMT and in inhibiting apoptosis (Wai melanomas (Figure 1a) and in the same set of samples, and Kuo, 2004; Chakraborty et al., 2006; Alonso et al., DNAJB6 expression was compromised (Figure 1b). Of 2007; Saika et al., 2007; Tuck et al., 2007; Chiodoni et al., those tumor specimens that expressed lower DNAJB6 2010). Elevated levels of OPN are associated with poor than normal controls, about 70% expressed higher OPN outcome in a variety of cancers such as breast cancer, than normal controls (exact binomial test, P ¼ 0.0032). mesothelioma, colon cancer and ovarian cancer (Furger These observations strongly imply an inverse relation- et al., 2001; Yeatman and Chambers, 2003; Greillier et al., ship between expression of DNAJB6 and OPN in 2008; Shih Ie and Davidson, 2009). In this study, we metastatic melanomas and prompted us to study the assessed DNAJB6 and OPN expression from melanoma mechanism of regulation of OPN by DNAJB6. Inde- specimens and found an inverse relationship between the pendently, estimation of cellular levels of DNAJB6 and transcript levels of these two genes. Here, we present that OPN from a panel of melanoma cells with increasing the J domain of DNAJB6 has a critical role in mediating malignant potential in comparison with normal human its activity. Specifically, the interaction of DNAJB6 with epidermal melanocytes revealed an inverse pattern of HSPA8 (heat-shock cognate protein, HSC70) is mediated expression and corroborated with observations made via the J domain and is responsible for the protein from melanoma specimens (Supplementary Figure 1). phosphatase 2 (PP2A)-driven dephosphorylation of gly- cogen synthase kinase 3b (GSK3b)atSerine9.This The J domain of DNAJB6 is involved in mediating OPN dephosphorylation activates GSK3b causing downregula- suppression tion of TCF/LEF (T cell factor1/lymphoid enhancer The J domain of HSP40 proteins is critical in mediating factor1) signaling leading to reduced OPN expression. We many of their known functions. To investigate if the J demonstrate that this downregulation is one of the domain of DNAJB6 has a role in regulating OPN, we important mechanisms of DNAJB6-mediated inhibition generated two mutant DNAJB6 cDNA constructs. One of malignancy. capable of coding for DNAJB6 devoid of the J domain (referred to as DJ) and the other that introduced mutations in the most conserved HPD tri-peptide of Results the J domain, changing them to three alanines (AAA; referred to as HPDMUT) (Figure 2, schematic of Loss of DNAJB6 in aggressive melanoma is inversely DNAJB6). Serum-free conditioned media from the related with gain of OPN stable expressors of wild-type DNAJB6 in MDA-MB- Elevated levels of OPN in tumor tissue and serum are 435 cells (435-DNAJB6), the corresponding vector associated with increased invasion and metastasis of control (435-V), 435-DNAJB6-DJ or 435-DNAJB6- melanoma (Denhardt, 2005; Zhou et al., 2005; Buback HPDMUT were assessed for OPN levels. While serum- et al., 2009). We had previously noted a possible free conditioned media from 435-V showed copious negative regulatory effect of DNAJB6 on OPN tran- amounts of OPN, OPN was undetectable in 435-DNAJB6. script levels (Mitra et al., 2008). To directly test the However, 435-DNAJB6-DJ and 435-DNAJB6-HPDMUT clinical relevance of this observation in melanoma, we showed higher levels of OPN, suggesting that the ability * P < 0.05 * P < 0.01 * P < 0.05 * P < 0.05 * P < 0.01 0.70 0.9 * P < 0.05 0.65 0.8 0.60 0.7 DNAJB6 0.55 0.6 levels of OPN Relative transcript 0.50 0.5 Relative transcript levels of Normal Normal Stage III Stage IV Stage III Stage IV Stage III A/B Stage III A/B Figure 1 Expression analysis of OPN and DNAJB6 in melanoma specimens. The Tissue Scan Melanoma qPCR Arrays (Origene Technologies) were queried using primer probes for (a) DNAJB6 and (b) OPN. Levels were normalized to human b-actin. The analysis includes 6 normal, 9 stage III, 6 stage IIIA/B and 22 stage IV specimens. *Indicates statistically significant differences. Oncogene DNAJB6 multiprotein complex with GSK3b regulates OPN A Mitra et al 4474 MDA-MB-435 A375 Schematic of DNAJB6 protein HPD DNAJB6 1 326 J OPN (SFM) DNAJB6-ΔJ 70 326 HPD-AAA DNAJB6 DNAJB6-HPDMUT DNAJB6-ΔJ 1 326 β-tubulin * P < 0.001 * P < 0.01 100 150 * P < 0.001 P < 0.001 P < 0.05 * 75 * * 100 P<0.001 50 expression expression 50 Percent OPN 25 Percent OPN 0 0 v DNAJB6ΔJ HPDMUT v DNAJB6 ΔJ HPDMUT Figure 2 Deletion of the J domain renders DNAJB6 incapable of suppressing OPN.