Differential roles of RET isoforms in medullary and papillary thyroid carcinomas

  1. Lois M Mulligan1,2⇑
  1. 1Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, Ontario, Canada
  2. 2Department of Pathology & Molecular Medicine, Queen’s University, Kingston, Ontario, Canada
  3. 3Michael Smith Genome Sciences Centre, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
  4. 4Department of Medical Genetics, University of British Columbia, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
  5. 5Department of Surgery, St Paul’s Hospital & University of British Columbia, Vancouver, British Columbia, Canada
  1. Correspondence should be addressed to L M Mulligan; Email: mulligal{at}queensu.ca
  1. View larger version:
    Figure 1

    RET isoform knockdown alters cell morphology and growth of thyroid carcinoma cell lines. (A) RET isoform expression in the PTC cell line TPC-1 and MTC cell line TT was quantified by real-time PCR using isoform-specific primers. Expression was normalized to the housekeeping gene GUSB. (B) Expression of total RET or individual RET isoform proteins in TPC-1 and TT cells. Whole cell lysates of MockKD and knockdown (KD) cell lines depleted for RET51 (RET9only), RET9 (RET51only) or total-RET KD (noRET) were immunoblotted using antibodies specific for each RET isoform or a panRET antibody (total RET) that detects both isoforms, as indicated. Protein was not obtained from noRET TT cells (not shown) as these cells were not viable and did not proliferate sufficiently for analysis. In TT cells, full-length RET appears as a doublet of immature and fully glycosylated forms. The chimeric RET/PTC1 in TPC-1 appears as a single band representing cytosolic protein. Tubulin serves as a loading control. (C) RET KD induces isoform-specific changes in TPC-1 and TT cell morphology. Phase contrast images (40×) showing morphology of the indicated RET KD cells. Scale bar = 100 µm. (D) Growth of TPC-1 and TT RET KD cells. Cells were transduced with lentiviral shRNA constructs (day 0) and growth was monitored over time as indicated. Representative images show cell and colony morphology at indicated times. (E) Quantification of proliferation of RET KD cells. Growth of indicated cells was measured by MTT assay after 72-h incubation. Data are shown as mean ± s.e.m. of three experiments, each with a minimum of three technical replicates, normalized to MockKD. In TPC-1 cells, RET51only, RET9only and noRET cell lines all proliferated significantly less than MockKD (*P < 2.4 × 10−9) and were each significantly different from each other (RET51only vs RET9only †P = 4.4 × 10−9; RET9only vs noRET ‡P = 3.0 × 10−9). RET51only and RET9only TT cells proliferated less than MockKD cells (*P < 3.0 × 10−5) but were not significantly different from each other (P = 0.20).

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    Figure 2

    RET knockdown induces isoform-specific changes in TPC-1 cell motility. Mean squared displacement (MSD) analysis of indicated RET KD TPC-1 cells seeded at (A) low or (B) high densities. Displacement from initial position was tracked at 45-min intervals over 20 h for a minimum of 100 individual cells/indicated condition. MSD for all tracked cells ± s.e.m. is plotted for each time point (left). Individual paths taken by 30 randomly selected cells are shown for each condition (right). MSD of noRET cells was reduced relative to MockKD at both low and high cell densities (P = 2.1 × 10−6 and 5.6 × 10−7, respectively). RET9only MSD was significantly lower compared to MockKD at high cell densities (P = 1.2 × 10−2) but not at low cell density (P = 0.42). (C) Representative phase-contrast images of the indicated TPC-1 cells migrating into a scratched wound in a confluent cell monolayer. Remaining wound area at the indicated time is highlighted by hatched lines. Wound closure was quantified after 18 h (right). RET9only and noRET TPC-1 cell wound closure was significantly reduced relative to MockKD control (*P = 6.3 × 10−3 and 3.7 × 10−5, respectively).

  3. View larger version:
    Figure 3

    RET knockdown alters the expression of epithelial and mesenchymal markers in TPC-1 and TT cells. (A and B) Expression of epithelial and mesenchymal marker transcripts quantified by qRT-PCR in the indicated RET KD (A) TPC-1 and (B) TT cells. Expression was normalized to expression of GUSB and expressed as fold change (2−ddCt) relative to MockKD. (A) RET KD in TPC-1 cells did not affect expression of SNAI1. However, expression of mesenchymal marker ZEB1 was reduced in all RET KDs (*P < 0.043) with RET9only (†P = 0.001) and noRET (‡P = 0.006) cells showing significantly greater reductions than RET51only cells. Expression of TWIST1 was reduced in RET9only and noRET cells (*P < 0.0023). CDH1 was below detection threshold in all cell lines except noRET. CDH2 expression was reduced by RET KD (*P < 0.023), with expression in RET9only and noRET cells significantly decreased relative to either MockKD or RET51only cells (†P < 7.6 × 10−6). (B) RET KD in TT cells did not affect SNAI1 expression, whereas ZEB1 expression was significantly reduced in both RET9only and RET51only cells (*P < 3.3 × 10−6). TWIST1 and CDH1 expression was undetectable in all conditions, whereas CDH2 expression was significantly reduced only in RET9only cells (*P = 7.1 × 10−5). (C and D) Immunofluorescence localization of (C) E-cadherin (red), RET (green) and (D) N-cadherin (green) in TPC-1 cells. (C) Arrows indicate accumulations of membranous E-cadherin in noRET cells. (D) Concentrations of N-cadherin (arrows) are visible at cell–cell contacts in MockKD, RET51only and RET9only cells but not in noRET cells. Nuclei are stained with Hoescht.

  4. View larger version:
    Figure 4

    RET knockdown reduces matrix metalloproteinase expression and secretion. (A) Quantification of MMP9 and MMP2 transcripts by qRT-PCR in the indicated RET KD TPC-1 (left) and TT (right) cells. Expression was normalized to GUSB mRNA expression level and expressed as fold change (2−ddCt) relative to MockKD. MMP9 expression was significantly reduced in RET9only and noRET TPC-1 cells compared to MockKD (*P < 5.2 × 10−6) and RET51only TPC-1 (†P < 9.5 × 10−12). MMP2 expression was reduced in all TPC-1 RET KD cells compared to MockKD (*P < 6.0 × 10−4) and in RET9only and noRET cells compared to RET51only TPC-1 (†P < 5.3 × 10−3). MMP9 expression was not detected in TT cells, but MMP2 expression was significantly reduced in RET9only TT cells relative to either MockKD or RET51only TT cells (*P < 1.2 × 10−9). (B) Representative images showing secretion of MMPs by RETKD TPC-1 and TT cells detected using gelatin zymography. Immature, secreted pro-MMP9 and pro-MMP2 are indicated by black arrowheads. The mature active MMP2 is indicated with a gray arrowhead. 1% FBS served as a positive control for enzymatic activity (gelatin degradation). (C) Bar graphs show the intensity of MMP bands in TPC-1 samples as shown in (B) quantified by densitometry and expressed relative to the corresponding pro-MMP band in the FBS control sample. Secretion of pro-MMP9 (left) was significantly reduced in all KD TPC-1 cell lines compared to MockKD cells (*P < 2.5 × 10−3) and reduced in RET9only and noRET cells relative to RET51only cells (†P < 5.8 × 10−3). Secretion of pro-MMP2 (centre) was significantly reduced in RET9only and noRET TPC-1 cells compared to MockKD (*P < 1.7 × 10−5) and RET51only (†P < 3.3 × 10−4) cells. Secretion of mature active MMP2 (right) by RET9only and noRET TPC-1 cells was significantly lower than MockKD (*P < 5.6 × 10−4) or RET51only (†P < 5.5 × 10−4) cells.

  5. View larger version:
    Figure 5

    RET knockdown restores TPC-1 contact inhibition of growth and reduces anchorage-independent growth of TPC-1 and TT cells. (A) Evaluation of contact inhibition of growth in RET KD cell lines. TPC-1 cells (left) and TT cells (right) were seeded at 1 × 105 cells/well and counted over indicated days to generate growth curves. Representative images of super-confluent layers of TPC-1 cells (day 7) and TT cells (day 30) are shown. (B) RET KD reduces anchorage-independent growth. TPC-1 cells (left) and TT cells (right) embedded in 30 mg/mL methylcellulose after 2-week incubation. In both TPC-1 and TT cells, RET51only and RET9only cells formed significantly fewer (*P < 1.2 × 10−8) and smaller (*P < 1 × 10−4) spheroids relative to MockKD cells (lower panels). RET9only cells of either cell line also formed significantly fewer spheroids than RET51only cells (†P < 1.9 × 10−9) but were significantly different in size only for TT cells (†P = 9.6 × 10−4). noRET TPC-1 cells did not form spheroids.

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    Figure 6

    RET knockdown reduces cell invasion. (A and B) RET KD in both TPC-1 and TT cells reduces invasion into collagen matrix. Pre-formed spheroids of 1 × 103 TPC-1 or TT cells were embedded in 2.5 mg/mL collagen matrix and allowed to invade for the indicated times. (A) RET51only TPC-1 cells show a modest reduction in invasiveness relative to MockKD, whereas RET9only and noRET cells have dramatically reduced invasive potential and invade a significantly shorter distance away from the spheroid than either MockKD or RET51only (*P < 8.2 × 10−9). noRET cells travel significantly shorter distances relative to RET9only (†P = 4.5 × 10−5). (B) RET51only TT cells display a modest reduction in invasiveness relative to MockKD, whereas RET9only cells invade significantly shorter distances relative to both MockKD and RET51only cells (*P < 1.9 × 10−2).

  7. View larger version:
    Figure 7

    Immunostaining of thyroid tissues for RET isoform expression. (A) Representative IHC staining of the indicated thyroid tissues for RET9 or RET51. Insets show low magnification image of the corresponding TMA tissue core. (B) RET9 and RET51 expression in benign and malignant thyroid tissues. Staining intensity scores for RET9 and RET51 are shown for each tissue subtype (bottom) and represented as box plots (top). Whiskers represent the data within the 1.5 interquartile range, whereas boxes represent the first (25%) and third quartiles (75%) as well as the median (50%). Outliers are represented by data points (dots) beyond the interquartile range. Mean RET9 staining intensity was significantly greater only in PTCs relative to benign tissues (*P < 0.001), while mean RET51 staining intensity was significantly greater in both PTC and MTCs relative to all other subtypes (†P < 0.001), RET51 staining was significantly stronger than RET9 only in PTCs (‡P = 0.003).

  8. View larger version:
    Figure 8

    RET functions in thyroid carcinoma cells. Localization and functions of RET9 and RET51 in MTC (left) and PTC (right) cells. Full-length RET9 (green) and RET51 (yellow) are shown at the cell membrane, whereas RET/PTC RET9 (green/purple) and RET51 (yellow/purple) localize in the cytosol. RET-associated processes common to MTC and PTC are indicated in green. RET-mediated processes occurring primarily in MTC (red) or in PTC (purple) are indicated. Arrow width is indicative of relative contributions of each RET isoform to the indicated cellular process.

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  1. doi: 10.1530/ERC-16-0393 Endocr Relat Cancer 24 53-69
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