Rapalog resistance is associated with mesenchymal-type changes in Tsc2-null cells

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

2 or on autoradiography film (USA Scientific 1968-3810), which was developed in an SRX-101A Film Processor (Konica) and then scanned.
After electrophoresis, gels were incubated in Novex Zymogram Renaturing Buffer (LC2670, Invitrogen) for 30 min at room temperature followed by a short incubation in Novex Zymogram Developing Buffer (LC2671, Invitrogen) for 30 min at room temperature and a longer incubation for 18 h at 37°C. Gels were washed 3 times with dH2O for 5 min at room temperature and stained with SimplyBlue SafeStain (LC6060, Invitrogen) for 2 hours at room temperature. Finally, gels were destained in dH2O for 2 h at room temperature, and images were acquired on a Foto/Analyst FX 6-7206.
For band intensity quantification, the ImageJ (version 1.52g) gel analysis tool was used on raw (unedited) images.

S.1.3. Xenograft studies.
Mice were inoculated with 0.1 ml cell suspension of ELT3 or ELT3-245 cells in PBS (2x10 7 per ml) by subcutaneous injection in each of both flanks. After palpable tumors formed, tumor width (W) and length (L) were measured twice per week using digital calipers. Tumor volume (V) was calculated using the formula [V = (W 2 × L) / 2] 2 . For treatments, rapamycin stock solution was prepared by dissolving 15 mg rapamycin (Selleck Chemicals S1039) in 1 ml DMSO and stored aliquoted at -20°C. Injectable rapamycin solution (0.6 mg/ml) was freshly prepared by dissolving 40 µl of the 15 mg/ml stock solution in 960 µl sterile normal saline (Becton Dickinson) containing 0.25% v/v Tween-80 (Sigma P8074) and 0.25% w/v PEG300 (Sigma 202371), or in 960 µl filter-sterilized corn oil (Sigma C8267). Mice were treated with rapamycin (3 mg/kg ip injection, 3 times per week) or vehicle control. Administration volume was 5 ml/kg body weight. At endpoint, mice were euthanized by CO2 narcosis followed by thoracotomy and removal of the heart and lungs. Whole blood was collected by cardiac puncture of anesthetized animals into EDTA-Na2 anticoagulant tubes.
Primary tumors were divided in equal parts, which were stored in RNAlater Stabilizing Solution (Thermo Fisher AM7021), or fixed in 10% neutral buffered formalin (Fisher Scientific), or snap-frozen in liquid nitrogen then stored at -80°C. All other organs/tissues were fixed in 10% neutral buffered formalin and processed to paraffin embedding and sectioning [University of Tennessee Health Sciences Center (UTHSC) Research Histology Core].
S.1.4. Establishment of cell lines from xenograft tumors. The skin of euthanized animals was disinfected with 70% ethanol, and the tumors were aseptically removed. For tissue dissociation, all media were supplemented with 10 µg/ml ciprofloxacin HCl (Bioworld 403100311). A portion of the tumor was placed in a tissue culture plate in a small volume of serum-free (SF) IIA complete and dissociated to < 1 mm 3 pieces by mechanical disruption with sterile scalpels. 5 ml of filter-sterilized 0.2% w/v collagenase type II (Worthington Biochemical Corporation LS004205) in SF IIA complete was added, and the tissue pieces were transferred into a 50 ml polypropylene tube. The tissue was washed three times with 0.2% w/v collagenase / SF IIA complete, resuspended in 5 ml 0.2% w/v collagenase / SF IIA complete and incubated at 37°C for 1-2 hours. Cells and tissue pieces were pelleted by centrifugation at 1,000 rpm for 10 min, and washed three times with 30 ml SF IIA complete without collagenase. The pellet was resuspended in IIA complete media supplemented with 5 mM L-glutamine, 15% v/v fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml ciprofloxacin HCl, and cultured at 37°C in a humidified atmosphere containing 5% CO2. Ciprofloxacin HCl was added to culture media during the first 10 passages of cells. S.1.5. Short-term lung colonization studies. Luciferase-expressing ELT3-245 cells were generated after transfection with phCMV-CLUC (Genlantis P003500) and selection of individual clones in 1 mg/ml G418 (Invitrogen) for 2 weeks.
Luciferase expression was assayed after cell lysis in Cell Culture Lysis Reagent (Promega E1531) using the Luciferase Assay System (Promega E1500) according to the manufacturer instructions. Relative luminescence was measured in a Synergy H1 multi-mode plate reader (Biotek). Luciferase-expressing ELT3 cells (ERL4) 3 were used as controls. Eightweek-old female CB17 SCID (The Jackson Laboratory, B6.CB17-Prkdcscid/SzJ) were pre-treated with a single dose of rapamycin (3 mg/kg ip), or vehicle control, for 48 hours. ERL4 and ELT3-245-luc-7 cells were pre-treated with 20 nM rapamycin or vehicle for 16 hours prior to intravenous inoculation in mice (2x10 5 cells in 0.1 ml PBS per mouse). At the time of inoculation, mice were treated with a second dose of rapamycin (or vehicle). Bioluminescence was measured after ip injection of XenoLight D-Luciferin (PerkinElmer 122799) using the Xenogen IVIS Spectrum In Vivo Imaging System (PerkinElmer) at 1 h, 6 h and 24 h post cell inoculation. Of note, we excluded animals that received poor luciferin administration during bioluminescence imaging. S.1.6. Sequencing of Mtor FKBP12-rapamycin binding domain coding regions. NCBI Primer-BLAST was used to design intronic primers spanning each of exons 41-46 of rat Mtor (Frap1) using rat chromosome 5 genomic sequence as reference (accession number NC_005104.4) (Supplementary Table 2). Genomic DNA was extracted from exponentially growing ELT3 and ELT3-245 cells using DNeasy Blood & Tissue Kit (Qiagen 69504), and was quantified in a Synergy H1 plate reader using Take3 microvolume plate (Biotek). PCR was performed using Platinum Hot Start (Invitrogen 13000012) on a Veriti Fast 96-well thermal cycler (Applied Biosystems). Amplicons were analyzed on a 2% w/v agarose gel in 1x TAE buffer, and purified with Purelink PCR purification kit (Invitrogen K310001). Sanger sequencing was conducted by UTHSC Molecular Resource Center. Reactions were analyzed on an ABI Prism 3130XL Genetic Analyzer (Applied Biosystems). Sequences from both ELT3 and ELT3-245 were aligned to the corresponding sequences from the rat genome (sequence assembly Rnor_6.0) using Jalview 2.10.4b1.
S.1.7. Gene expression studies. Total RNA was isolated from exponentially growing cell cultures using RNeasy Mini kit (Qiagen 74104). RNA was quantified in a Synergy H1 multi-mode plate reader using a Take3 microvolume plate (Biotek). Samples with OD260/OD230 < 1.8 were subjected to linear acrylamide / ethanol precipitation to increase purity.
Microarray gene expression assays were conducted by the UTHSC Molecular Resource Center. RNA integrity was assessed on a 2100 Bioanalyzer System using an RNA 6000 Nano Kit (Agilent). Assays were performed from 1 µg total RNA on Clariom S rat-specific arrays (Affymetrix). Arrays were scanned on a GeneChip Scanner (Affymetrix).
Microarray gene expression data analysis was conducted by the UTHSC Molecular Bioinformatics Core. Fold-change (FC) for gene expression was calculated using standard methods. A FC threshold of 1.5 was applied to identify differentially expressed genes. A Welch t test and Benjamini -Hochberg false discover rate were used to calculate statistical significance. Genes with an adjusted p-value ≤ 0.05 where considered statistically significant. Pathway analyses were performed using iPathwayGuide (Advaita).
For gene expression studies by RT-qPCR, the SuperScript VILO cDNA Synthesis Kit (Invitrogen 11754050) was used to synthesize cDNA from 1 µg total RNA in a reaction volume of 20 µl. The cDNA synthesis reaction was diluted 1:4 with nuclease-free H2O (Ambion AM9930), and 2 µl of diluted cDNA was used for target amplification using the TaqMan Fast Advanced Master Mix (Applied Biosystems 4444557) and gene-specific TaqMan assays (Supplementary Table 3) in technical triplicates. Actb (b-actin) was used as housekeeping gene. Amplification and detection were 5 performed in an ABI 7500 Fast Real-Time PCR System. Pairwise fold-change (FC) differences were calculated using the DDCt (Livak) method 4,5 . FC values less than 0.5 or greater than 1.5 were considered significant.        Fig. 4A. (continued) The rabbit E-cadherin antibody (CST #3195) is not crossreactive for rat E-cadherin. The mouse E-cadherin antibody (CST #14472) is cross-reactive for rat E-cadherin.    Fig. 5D. Digital photographs of zymogram gel for MMP2. Raw (unedited) image is shown on the left. Levels-adjusted image is shown on the right. The black dashed-line rectangle corresponds to the cropped gel section shown in Fig. 3F. Figure S15. KEGG pathway map (with pathway analysis) and gene expression differences for ErbB signaling between rapamycin-treated ELT3-245 vs ELT3. Genes with negative logFC values (marked in blue) are up-regulated. Genes with positive logFC values (marked in red) are down-regulated. Figure S16. Heat map of ErbB signaling genes that were differentially expressed between rapamycin treated ELT3-245, compared to rapamycin treated ELT3. Figure S17. KEGG pathway map (with pathway analysis) and gene expression differences for ECM-receptor interaction signaling between rapamycin-treated ELT3-245 vs ELT3. Genes with negative logFC values (marked in blue) are upregulated. Genes with positive logFC values (marked in red) are down-regulated. Figure S18. Heat map of integrin genes that were differentially expressed between rapamycin treated ELT3-245, compared to rapamycin treated ELT3.