Osteoblast-Secreted Factors Mediate Dormancy of Metastatic Prostate Cancer in the Bone via Activation of the TGFβRIII-p38MAPK-pS249/T252RB Pathway.

Cell lines, antibodies and reagents Cell lines: Human PCa C4-2B4 (gift from Robert Sikes, University of Delaware, 2006) ( 3 , 4 ), C4-2b (gift from Leland Chung, Cedars-Sinai Medical Center, 2004) ( 5 ), PC3-mm2 (gift from Isaiah J. Fidler, M.D. Anderson Cancer Center, 2005), 22Rv1 (ATCC, 2015) ( 6 , 7 ), and BPH-1 (gift from Simon Hayward, Vanderbilt University, 2015); mouse osteoblast precursor MC3T3 (ATCC, 2006); human lung carcinoma A549 (ATCC, 2013). The identity of all cell lines was verified by polymorphic Short Tandem Repeat loci (STR) profiling, and all cell lines are mycoplasma free. Recombinant human proteins: TGFβ2, TGFβ1, GDF10 (BMP-3b) (R&D). Antibodies: phospho-p38MAPK, p38MAPK, RB, Smad2/3, phospho-Smad1/5, p27Kip1, GAPDH, β-actin (Cell Signaling); phospho(S249/T252)-RB ( 8 ); Ki-67 (Dako); TGFβ2 and TGFβRIII (Proteintech); GDF10, p21 and TGFβRIII (R&D); β-tubulin, α-tubulin (GeneTex). Reagents: mouse TGFβ2 (Genorise) and osteocalcin (Alfa Aesar) ELISA kits; p38MAPK inhibitor SB202190 (InvivoGen); Vybrant DiO lipophilic membrane dye (Invitrogen). Subcutaneous, intra-femural, and intracardiac injection of PCa cells Luciferase-expressing PCa cells were injected under the skin, into the femurs ( 9 ), or into the left ventricle ( 10 ) of male SCID mice. Tumor growth was monitored using bioluminescence imaging. Animal studies were performed in accordance with as well as approved by an Institutional Animal Care and Use Committee (IACUC).

Immunohistochemistry

Tumor bearing femurs were fixed in formaldehyde and decalcified with formic acid before embedded in paraffin. Immunohistochemistry was performed as described ( 11 ). Differentiation of primary mouse osteoblasts Primary mouse osteoblasts (PMOs) were isolated from calvariae of 2-5 day-old pups, cultured to confluence, and the medium switched to differentiation medium, which contains ascorbic acid and β-glycerol phosphate. Osteoblast conditioned medium (OB-CM) was concentrated and medium exchanged by Centricon (Millipore) centrifugation.

Gene array analysis

RNAs prepared from undifferentiated and differentiated PMOs were subjected to whole-genome microarray analysis (4×44K, Agilent Technologies) (Arraystar, Inc). Array data are deposited in NCBI GEO under accession number GSE90127 . PCR RNAs from PMOs were analyzed by qRT-PCR using mouse primers ( Supplementary Table S1 ). Total DNA was prepared from mouse hind legs and used for real-time PCR using primers for human Alu repetitive sequences ( Supplementary Table S1 ). The number of tumor cells in bone was calculated based on Alu PCR of a serial dilution of DNA from C4-2B4 cells.

Live-cell time-lapse imaging

PCa cells were plated in Hi-Q4 dishes (Ibidi) and cultured in RPMI-1650 containing 1:20 dilution of Day 0, 6, 24 or 30 OB-CM or in RPMI-1640 containing 0.1% FBS with TGFβ2, GDF10 or TGFβ1. Images were acquired every 20 min for 72 h in a BioStation (Nikon). Grid-500 glass-bottom dishes (Ibidi) were used for live-cell monitoring in more fields. Data were compiled using NIS-Elements (Nikon) software.

Immunofluorescence imaging

Following live-cell imaging, cells were fixed and permeabilized, co-incubated with anti-Ki67 and anti-p27, and re-imaged on the BioStation. Proximity ligation assay (PLA) Proximity ligation assay was performed using Duolink PLA In Situ Green Starter Kit (Mouse DUO92004/Rabbit DUO92004, Sigma). Primary antibodies were anti-phospho-p38MAPK (28B10) and anti-phospho-(S249/T252)-RB ( 8 ). Images were acquired using FluoView 1000 IX2 confocal microscopy (Olympus).

Generation of C4-2B4 cells with knockdown of TGFβRIII

TGFβRIII was knocked down by RNA interference via lentivirus-expressing shRNAs in pGIPZ. Clones C4-2B4-pGIPZ-sh-TβRIII #2 and #3 were generated. Antisense sequences for sh-TβRIII Clone #2: 5′-ATAGCTCCATGTTGAAGGT-3′ ( NM_001195683 ) and Clone #3: 5′-ATAGTAGACCACACCATCA-3′ (MN_003243).

Generation of C4-2B4 and C4-2b cells with dominant-negative p38MAPK

C4-2B4 and C4-2b cells were transduced with retroviral-expressing p38α dominant-negative MAPK (p38DN), containing mutations in the activation loop between the two kinase domains, from Thr180-Gly-Tyr182 to Ala180-Gly-Phe182 ( 12 ), in a pBMN-I-GFP vector. Human PCa datasets The Kaplan-Meier method with log-rank test was used to evaluate overall disease-specific survival curves for PCa patients from the Nakagawa dataset ( 13 ). Patients from the Taylor data set ( 14 ) were used to evaluate PCa metastasis. Patients from the Lapointe dataset ( 15 ) were used to evaluate PCa metastatic progression. For computing TGFBR3 gene score based on expression profiling data from human PCa tumors, TGFBR3 gene was first z -normalized to SD from the median across the primary tumor samples. The average of the z -normalized values was used to represent the score for each sample profile.

Statistical analysis

Data quantification was performed by using the Student’s t -test and expressed as mean ± s.e.m. P values of < 0.05 were considered statistically significant.

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