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

Abstract

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.

Figure 1

ELT3-245 are resistant to rapamycin. (A) Female ovariectomized CB17/SCID mice were inoculated subcutaneously with ELT3 cells. After tumor development, mice were randomized in treatment cohorts of vehicle (n = 27, circles, black line), or rapamycin (3 mg/kg ip three times a week, n = 19, red lines). A single tumor from mouse #245 in the rapamycin cohort (upward triangles) did not respond to treatment. (B) ELT3-245 cells have enhanced spindle-like characteristics, compared to ELT3. Scale is 250 μm. (C) Rapamycin has no significant effect on ELT3-245 growth. Equal numbers of ELT3 and ELT3-245 cells (n = 3) were cultured for 4 days in the presence of DMSO or rapamycin, and living cells were counted using trypan blue exclusion. (D) ELT3-245 cells have delayed dephosphorylation of ribosomal protein S6 upon rapamycin treatment, compared to ELT3. Cells were incubated with 2 nM rapamycin for the indicated time. Ratios of p-S6: Total S6 are normalized to the 0 h time point value within each group. Full-length blots are presented in Supplementary Fig. 3. (E) Mice inoculated with ELT3-245 cells develop tumors faster, compared to ELT3. Non-ovariectomized female CB17/SCID mice were inoculated subcutaneously with equal number of ELT3 or ELT3-245 cells (n = 26 per group). ELT3 and ELT3-245 tumor-bearing mice were monitored daily for formation of palpable tumors. (F) ELT3-245 tumors do not respond to rapamycin treatment in vivo. ELT3-245 tumors partially, but not statistically significantly, responded to rapamycin at day 8, however they significantly grew beyond baseline by day 22. ELT3 tumors rapidly responded to rapamycin treatment by day 8, and the response was sustained until day 22. Vehicle-treated tumors exceeded 4,000 mm³ before day 22, and mice that reached end-point criteria were removed from the study and euthanized.

Figure 2

ELT3-245 cells exhibit enhanced anchorage-independent cell growth and resistance to anoikis. (A) Automatic particle analysis of colonies from soft agar assay. ELT3-245 cells formed significantly bigger colonies, compared to ELT3 cells (****P < 0.0001). (B) 24 hours after plating ELT3 and ELT3-245 cells in soft agar, cultures were continuously treated with 20 nM rapamycin for 3 weeks. Rapamycin treatment did not cause a significant reduction in colony volume in ELT3-245 cells, compared to DMSO. However, rapamycin caused a significant increase in colony volume in ELT3 cells (*P = 0.0148). (C) A subset of ELT3 cells are rapamycin-resistant. Volume distribution of DMSO- and rapamycin-treated parental ELT3 in soft agar. Approximately 19% of rapamycin-treated ELT3 colonies were bigger than DMSO-treated ELT3 colonies. (D) The top 19% of rapamycin-treated ELT3 colonies were significantly bigger, compared to the bottom 81% of rapamycin-treated ELT3 colonies (P < 0.0001). The top 19% of rapamycin-treated ELT3 colonies were not significantly bigger, compared to DMSO-treated ELT3-245 colonies (P > 0.05). (E) ELT3 and ELT3-245 cells were grown in non-adherent condition for 6 and 24 hours, harvested and the percentage of dead cells was counted by trypan blue exclusion. 0 h indicates percentage of dead cells immediately after trypsinization and resuspension in growth media. (F) Immunoblotting of lysates from cells grown in suspension conditions for 0, 6 and 24 hours from panel E. Full-length blots are presented in Supplementary Fig. 6.

Figure 3

ELT3-245 cells metastasize to the lungs. (A) Micrographs (10x objective) of hematoxylin-eosin stained sections from lungs of ELT3-245 (a,b) and ELT3 (c,d) tumor-bearing mice treated with vehicle (a,c) or rapamycin (b,d). PV = pulmonary vein. Arrow in panel b indicates invasion of ELT3-245 cells through basal membrane. Arrows in panel d indicate the location of ELT3 micro-metastases. (B) a Bioluminescence images of vehicle-treated SCID mice that were inoculated with 2 × 10⁵ luciferase-expressing ELT3 or ELT3-245 cells pre-treated with DMSO for 16 hours. Mice (n = 4 per group) were imaged at 1 h (baseline), 6 h and 24 h post-inoculation. b Plot of the relative lung colonization (photon flux normalized to baseline) for the mice shown in panel a. *Indicates P < 0.05. Error bars are SEM.

Figure 4

ELT3-245 cells exhibit mesenchymal signaling characteristics. (A) EMT marker immunoblotting of ELT3 and ELT3-245 lysates from untreated cells or cells treated with 100 nM rapamycin for 3 days. Full-length blots are presented in Supplementary Fig. 9. (B) Heat map of epithelial marker associated genes that were differentially expressed between rapamycin treated ELT3-245, compared to rapamycin treated ELT3. (C) Nuclear and cytoplasmic fractions of ELT3 and ELT3-245 cells treated with 100 nM rapamycin (or DMSO) for 3 days were immunoblotted for β-catenin. Full-length blots are presented in Supplementary Fig. 10. (D) Heat map of β-catenin target and Wnt signaling genes that were differentially expressed between rapamycin treated ELT3-245, compared to rapamycin treated ELT3.

Figure 5

Gene expression changes associated with ELT3-245 cells. (A) Gene expression analysis of upregulated (left) and downregulated (right) genes in ELT3-245 cells vs ELT3 cells under vehicle treatment (DMSO, gray bars, closed circles) or rapamycin treatment (20 nM for 24 hours, white bars, open squares). The 0.5 < FC < 1.5 thresholds for significant gene expression differences are indicated with red horizontal lines. (B) Effect of rapamycin treatment vs DMSO treatment on gene expression in ELT3 (gray bars, closed circles) and ELT3-245 cells (white bars, open squares). (C,D) ELT3 and ELT3-245 cells were cultured in serum-free media for 24 or 48 hours, in the presence of 100 nM rapamycin (or DMSO). (C) Lysates were immunoblotted for MMP2. Full-length blots are presented in Supplementary Fig. 13. (D) Media from cell cultures were collected and analyzed by zymography for MMP2 activity. Numbers below the zymogram gels show relative activity of MMP2 compared to DMSO-treated ELT3 cells. Full-length gel is presented in Supplementary Fig. 14.