Online Supplementary Information

ONLINE SUPPLEMENTARY INFORMATION

Restored expression of the atypical heat shock protein H11/HspB8 inhibits tumor growth through activation of different TAK1-dependent death pathways in genetically diverse melanoma cells

Cynthia C. Smithǂ, 1, Ki Seok Leeǂ, 1, Baiquan Liǂ, Jennifer M. Laingǂ, J Herslǂ, Marianna Shvartsbeyn§, and Laure Aurelianǂ, 2

ǂDepartment of Pharmacology, University of Maryland, School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201, USA. §Department of Pathology, New York University, 522 First Avenue, Smilow, 301C, New York, NY 10016

1Cynthia C. Smith and Ki Seok Lee contributed equally to this research 2To whom correspondence should be addressed: Laure Aurelian, Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201-1559, Tel: 410-706-3895; Fax: 410-706-2513; E-mail: [email protected]

SUPPLEMENTARY MATERIALS AND METHODS

Cells. Adult human melanocytes (NHM) were purchased from Cascade Biologicals (Portland,

Ore) and grown in medium 154 supplemented with 3ng/ml basic fibroblast growth factor, 0.3% bovine pituitary extract, 3 μg/ml heparin, 0.18 μg/ml hydrocortisone, 5 μg/ml insulin, 5 μg/ml transferring and

10nM endothelin-1 (human melanocyte supplement -2). Melanoma cells A375 and A2058 were obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbeco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS, Gemini Bioproducts, Calabasos, CA) 4.5 g/L glucose, 1500 mg/ml sodium bicarbonate, and 4 mM glutamine.

Tet–inducible H11/HspB8 retrovirus vector and stable cell transfection. The tet-inducible approach is widely used for targeted gene expression and pro-apoptotic cellular proteins placed under appropriate Doxycycline (Dox)-induced regulation through the use of tet-inducible promoters are an established conditional suicide mechanism 1-14. H11/HspB8 DNA (0.6kb) was obtained by EcoR1/XbaI digestion of p3XFlag-CMV-H11/HspB815. The Xba site was blunt ended and the fragment ligated into the

EcoRI/SmaI site of pIRES2-EGFP vector (Clontech). A fragment containing H11/HspB8-IRES-EGFP was isolated by EcoR1/NotI digestion, blunt ended and ligated into the HpaI site of the pRev-TRE

1 retroviral vector, in which the MMTV promoter drives a hygromycin resistance gene and the tet-sensitive promoter (tet response element upstream of minimal CMV IE promoter) drives H11/HspB8. To generate retroviruses, the PT57 packaging cell line was transfected with pRevTet-On (contains G418 resistance cassette) or pRevTRE-H11/HspB8 and Lipofectamine 2000. Virus titers were assayed as per manufacturer’s instructions. Melanoma cells were infected with the tet-On retrovirus at a multiplicity of infection (moi) of 50. Clones were selected with G418 (1mg/ml), infected with the TRE-H11/HspB8 retrovirus (moi = 50) and selected with 800µg/ml hygromycin. H11/HspB8 overload was induced with

Doxycycline (Dox; 2, 5 or 10µg/ml). The tet-inducible H11/HspB8 construct was previously described16

Antibodies. Antibodies to JNK, phosphorylated JNK (pJNK), Akt and phosphorylated (activated)

Akt (pAkt), were from Cell Signaling Technology (Danvers, MA). Antibodies to ERK1/2 , ASC and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), antibody to phosphorylated (activated)

ERK 1/2 (pERK1/2) from Promega (Madison, WI) and antibody to JIP-1 from Abcam (Cambridge, MA).

Immunoprecipitation and Immunoblotting. The preparation of protein extracts and their assay by immunoblotting or immunoprecipitation/immunoblotting was previously described15-18. Briefly, cultured cells were lysed with radioimmunoprecipitation buffer [RIPA; 20 mM Tris-HCl (pH 7.4), 0.15 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate] supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and sonicated twice for

30 seconds at 25% output power with a Sonicator ultrasonic processor (Misonix, Inc., Farmingdale, NY).

Xenograft tissues were weighed, resuspended in RIPA buffer (0.5ml/g), homogenized using a pre-chilled motorized pestle (Kontes, Vineland NJ) and cleared of cell debris by centrifugation (16,000g; 4C for

30min). Protein concentrations were determined by the bicinchoninic assay (Pierce, Rockford, IL).

Quantitation was done by densitometry using the Bio-Rad GS-700 imaging densitometer and pixel density was determined using Multi Analyst software (Bio-Rad, Hercules, CA). Results are expressed as mean densitometric units ± SD

SUPPLEMENTARY RESULTS

2 Untransfected A375 and A2058 have different levels of activated ERK and Akt that are not altered by Dox. ERK and Akt are frequently activated in melanoma cells. To determine whether ERK and Akt are differently activated (phosphorylated) in A2058 as compared to A375 cells and examine the potential effect of Dox treatment, protein extracts obtained form the untransfected cells that had been untreated or treated with Dox (5 g/ml; 3 days) were immunoblotted with antibody to pERK1/2, stripped and re-probed with antibody to total ERK1/2 and duplicate samples were similarly immunoblotted with antibodies to pAkt followed by total Akt. Extracts from normal melanocyte cultures were studied in parallel and served as control. The data summarized in Fig. S1A, indicate that the levels of activated

(phosphorylated) ERK and Akt are significantly higher in A375 and A2058 cells than in normal human melanocytes. However the levels of pERK1/2 are significantly higher in A375 than A2058 cells, while those of pAkt are slightly higher in A2058 than A375 cells. Dox did not alter Akt or ERK activation, with similar levels of pAkt and pERK1/2 seen in the untreated and Dox-treated cells.

Untransfected A375 and A2058 differ in the expression of JIP1 and ASC. The JNK scaffold protein JIP1 is expressed in A375, but not A2058 cells (Fig. S1B). By contrast, the apoptosis-associated speck-like protein containing a CARD (ASC) is expressed in A2058 but not A375 cells (Fig. S1C).

Collectively the data confirm independent conclusions that A2058 and A375 melanoma cells are molecularly distinct19,20.

H11/HspB8 activates JNK in transfected A375 but not A2058 cells. Protein extracts from

A375 and A2058 cells stably transfected with tet-inducible H11/HspB8 and treated or not with Dox

(5g/ml; 3 days) were immunoblotted with antibodies to H11/HspB8, pJNK and JNK and the data were quantified by densitometric scanning. H11/HspB8 was expressed equally well in both cell lines, but pJNK was only seen in A375 cells and its levels were increased by Dox treatment. JNK was minimally expressed in A2058 cells and it was not activated by Dox treatment, likely related to the absence of the scaffold protein JIP1, which is required for JNK activation21.

3 H11/HspB8 inhibits TRAF6/TAB2 binding in A2058 cells. The E-3 ligase TRAF6 binds the type I TGF- receptor and upon TGF- stimulation it activates TAK1. The exact role of polyubiquitination in TAK1 activation is still unclear. It has been suggested that it may promote dimerization and autophosphorylation of TAK1, or recruitment of TAB2 to the TAK1-TRAF6 complex22.

We have previously shown that TAK1 binds TAB1 in the presence or absence of H11/HspB816 , but the effect of H11/HspB8 on TAK1/TAB2 binding and/or binding of TAB2 to TRAF6 are unknown. To address these questions, extracts of stably transfected A2058 cells treated or not with Dox (5g/ml; 3 days) were assayed by imunoprecipitation/immunoblotting with TAB2 and TRAF6 antibodies. The data summarized in Fig. S3, indicate that H11/HspB8 had, at best, a minimal effect on TAK1/TAB2 binding but it inhibited the binding of TAB2 to TRAF6, suggesting that H11/HspB8 interferes with TRAF6 recruitment to TAK1 and its function in TAK1 polyubiquitination. This is consistent with our finding that

H11/HspB8 binds and activates TAK1 through direct phosphorylation (Manuscript Fig. 2).

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure S1 . Untransfected A375 and A2058 cells have different levels of pERK1/2, pAKT, JIP1 and ASC. A . Extracts from untransfected A2058 and A375 cells treated or not with Dox (5g/ml; 3 days) and normal human melanoctyes (NHM) were immunoblotted with antibodies to pERK1/2 or pAkt . The blots were stripped and re-probed with antibodies to total ERK1/2 or Akt. Data were quantified by densitometric scanning and the results are expressed as fold-activation

(pERK1/2/ERK1/2 or pAkt/Akt ratios for A375 and A2058 cells over the same ratios for NHM  SD. B.

Extracts from untransfected A2058 and A375 cells were immunoblotted with antibody to JIP, stripped and re-probed with antibody to GAPDH (loading control). C. Extracts from untransfected A2058 and

A375 cells were immunoblotted with antibody to ASC, stripped and re-probed with antibody to actin

(loading control).

Supplementary Fig. S2. H11/HspB8 does not activate JNK in stably transfected A2058 cells. Extracts from A2058 and A375 stably transfected with tet-inducible H11/HspB8 and cells treated or not with Dox (5

4 g/ml; 3 days) were immunoblotted with antibody to H11/HspB8. Blots were sequentially stripped and re-probed with antibodies to activated (phosphorylated) JNK (pJNKThre183/Tyr185) followed by total JNK. Data were quantified by densitometric scanning and the results are expressed as pJNK/JNK densitometric units  SD.

Supplementary Fig. S3 . H11/HspB8 inhibits TRAF6/TAB2 binding in stably transfected A2058 cells.

A. Extracts from A2058 cells stably transfected with tet-inducible H11/HspB8 and treated or not with Dox

(5g/ml; 3 days) were immunoblotted (IB) with antibody to TAB2 (Extract) or immunoprecipitated (IP) with antibody to TAK1, TAB2 or IgG (control) and immunoblotted with antibody to TAK1. B. Extracts from stably transfected A2058 cells as in A were either immunoblotted with antibody to TRAF6 (Extract) or immunoprecipitated with antibody to TAB2 or IgG (control) and immunoblotted with antibody to TRAF6.

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