Calcitonin induces stem cell-like phenotype in prostate cancer cells

in Endocrine-Related Cancer
Correspondence should be addressed to G V Shah: shah@ulm.edu
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Stem cell-like-cancer cells are key drivers of tumor growth, metastasis, and relapse of cancer following remission. Prostate stem cell-like cancer cells isolated from human prostate cancer (PC) biopsies express CD44+2β1 hi/CD133+ cell surface markers and can self-renew in vitro. Expression of calcitonin (CT) and its receptor (CTR) is frequently elevated in PCs and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We investigated whether CT-CTR autocrine axis induces stem cell-like phenotype in two PC cell lines. CT-CTR axis in these cell lines was activated by enforced expression of CTR. The cells were then examined for the changes in the expression of CD44 and CD133, collagen adherence, tumorigenic, metastatic and repopulating characteristics. The activation of CT-CTR axis led to a large increase in adherence to collagen and a remarkable increase of CD44 and CD133 in PC-3 and LNCaP cells. This was accompanied by a strong increase in tumorigenic, metastatic and repopulation properties of PC cells. However, the mutation of CTR-C PDZ-binding site in CTR almost abolished CTR-mediated increases in stem cell-like characteristics of PC cells. These results support an important role for CT-CTR axis in the progression of PC from localized cancer to an aggressive form, and a majority of proinvasive CTR actions may be mediated through its interaction with its partner protein at the PDZ-binding site. These results suggest that CT/CTR can serve as a valuable target to prevent the generation of stem-like PC cells.

Downloadable materials

  • 1. The representative dot plots of flow cytometric evaluation of CD44 expression in PC3v cell population and its corresponding CD44 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 2. The representative dot plots of flow cytometric evaluation of CD133 expression in PC3v cell population and its corresponding CD133 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 3. The representative dot plots of flow cytometric evaluation of 21 expression in PC3v cell population and its corresponding 21 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 4. The representative dot plots of flow cytometric evaluation of CD44 expression in PC3-CTRwt cell population and its corresponding CD44 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 5. The representative dot plots of flow cytometric evaluation of CD133 expression in PC3-CTRwt cell population and its corresponding CD133 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 6. The representative dot plots of flow cytometric evaluation of 21 expression in PC3-CTRwt cell population and its corresponding 21 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 7. The representative dot plots of flow cytometric evaluation of CD44 expression in PC3-CTRESS cell population and its corresponding CD44 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 8. The representative dot plots of flow cytometric evaluation of CD133 expression in PC3-CTRESS cell population and its corresponding CD133 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 9. The representative dot plots of flow cytometric evaluation of 21 expression in PC3-CTRESS cell population and its corresponding 21 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 10. The representative dot plots of flow cytometric evaluation of CD44 expression in LNCaP cell population and its corresponding CD44 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 11. The representative dot plots of flow cytometric evaluation of CD133 expression in LNCaP cell population and its corresponding CD133 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 12. The representative dot plots of flow cytometric evaluation of 21 expression in LNCaP cell population and its corresponding 21 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 13. The representative dot plots of flow cytometric evaluation of CD44 expression in LNCaP-CTRwt cell population and its corresponding CD44 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 14. The representative dot plots of flow cytometric evaluation of CD133 expression in LNCaP-CTRwt cell population and its corresponding CD133 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 15. The representative dot plots of flow cytometric evaluation of 21 expression in LNCaP-CTRwt cell population and its corresponding 21 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 16. The representative dot plots of flow cytometric evaluation of CD44 expression in LNCaP-CTRESS cell population and its corresponding CD44 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 17. The representative dot plots of flow cytometric evaluation of CD133 expression in LNCaP-CTRESS cell population and its corresponding CD133 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.
  • 18. The representative dot plots of flow cytometric evaluation of 21 expression in LNCaP-CTRESS cell population and its corresponding 21 positive percentile measurements in the control, fast adherent (5’min), slow adherent (20’min) and non-adherent (>20 min) cell populations. All the plots depict log fluorescence values of FITC.

 

      Society for Endocrinology

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Figures

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    Fractionation of PC-3 cells. (A) Experimental protocol for fractionation of PC-3 cells on the basis of collagen adherence. (B) Percentage of PC-3 cells separated from total cells separated in each stage of fractionation. The results are expressed as mean ± s.e.m. of percent of cells fractionated at each stage of eight separate experiments. (C) Percentage of LNCaP cells separated from total cells separated in each stage of fractionation. The results are expressed as mean ± s.e.m. of percent of cells fractionated at each stage of eight separate experiments. *P < 0.001 (one-way ANOVA and Newman–Keuls test (PC-3CTRwt-fast vs all other PC-3CTRwt subfractions)). (D1) CTR immunoreactivity in plasma membranes of PC-3 and LNCaP sublines expressing either CTR-wt or CTRΔESS (50 μg protein/lane) was determined by Western blot analysis. The blot was reprobed for ß-actin for normalization. Representative of three separate experiments. (D2) Optical density of CTR and actin bands on each blot was obtained by image analysis on Kodak 4000 MM Image station. The figure presents the group data (ratio of CTR OD/actin OD ± s.e.m. of three blots).

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    Co-expression of CD44/CD133 on surface of PC-3 cells. (A) Percentage of cells double-stained with CD44/CD133 antibodies. The results are mean ± s.e.m. of four separate experiments. (B) CD44/CD133 immunocytochemistry of PC-3 cell subfractions. Fast-, slow- and non-adherent fractions of PC-3, PC-3-CTRwt and PC-CTR-ΔESS cells were double-labeled for CD44 and CD133 as described in ‘Methods’ section. The slides were photographed and their IHC index was calculated. The results are mean ± s.e.m. of three separate experiments. **Significantly different from its corresponding PC-3 subfraction. Level of significance: P<0.001 (one-way ANOVA and Newman–Keuls test). For example, PC-3-CTRwt fast vs PC-3 fast; PC-3CTRwt slow vs PC-slow; PC-3CTRwt-non vs PC-3-non. ^Significantly different from PC-3 CTRwt fast or PC3-CTRwt-slow. Levels of significance: P < 0.01 (one-way ANOVA and Newman–Keuls test).

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    Rate of cell proliferation of PC-3 cell subfractions: Effect of CTR. Fast-, slow-and non-adherent fractions of PC-3, PC-3CTR and PC3-CTRΔESS cells were analyzed for cell proliferation by MTT assay as described in ‘Methods’ section. The results are expressed as mean ± s.e.m. of four separate experiments. *P<0.001 (one-Way ANOVA and Newman–Keuls test (PC-3CTRwt-fast vs all other PC-3CTRwt subfractions)).

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    Migration of PC-3 cell subfractions: Effect of CTR. Fast-, slow- and non-adherent fractions of PC-3, PC-3CTR and PC3-CTRΔESS cells were analyzed for cell migration in a Matrigel™ invasion assay as described in ‘Methods’ section. The results are presented as (A) representative micrographs as well as (B) pooled data of four separate experiments (mean ± s.e.m.). *P < 0.001 (one-way ANOVA and Newman–Keuls test (PC-3CTRwt-fast vs all other PC-3CTRwt subfractions)). A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0333.

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    Invasion of PC-3 cell subfractions: Effect of CTR. Fast-, slow-and non-adherent fractions of PC-3, PC-3CTR and PC3-CTRΔESS cells were analyzed for cell invasion in a wound healing assay. The results are presented as (A) representative micrographs as well as (B) pooled data of four separate experiments (mean ± s.e.m.). *P < 0.001 (one-way ANOVA and Newman–Keuls test (PC-3CTRwt-fast vs all other PC-3CTRwt subfractions)). A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0333.

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    Spheroid formation by PC-3 cell subfractions: Effect of CTR. Fast-, slow- and non-adherent fractions of PC-3, PC-3CTR and PC3-CTRΔESS cells were analyzed for their ability to form spheroid in defined conditions as described in ‘Methods’ section. The results are presented as (A) representative micrographs as well as (B) pooled data of four separate experiments (mean ± s.e.m.). *P < 0.001 (one-way ANOVA and Newman–Keuls test (PC-3CTRwt-fast vs all other PC-3CTRwt subfractions)). A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0333.

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    Clonogenicity of PC-3 cell subfractions: Effect of CTR. Fast-, slow- and non-adherent fractions of PC-3, PC-3CTR and PC3-CTRΔESS cells were analyzed for clonogenic activity. The results are presented as (A) representative micrographs as well as (B) pooled data of four separate experiments (mean ± s.e.m.). *P < 0.001 (one-way ANOVA and Newman–Keuls test (PC-3CTRwt-fast vs all other PC-3CTRwt subfractions)). A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0333.

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    Distant micrometastases in nude mice. (A) Representative photomicrographs of H&E stained tissues of nude mice (as labeled) implanted orthotopically with fast/slow adherent PC-3-CTR-wt and PC-3-CTRΔESS cells (Magnification 400×). Presence of micrometastases is indicated by arrows. (B) The presence of micrometastases in distant organs presented in Fig. 9A was confirmed by PSCA immunofluorescence. (C) Mean area of micrometastases was measured over total microscopic optical field (40×) for fast/slow adherent PC-3-CTR-wt and PC-3-CTRΔESS cells and represented as percentile of total area. *P < 0.05. Fast adherent (PC-3-CTR-wt vs PC-3-CTRΔESS); #P < 0.05 slow adherent (PC-3-CTR-wt vs PC-3-CTRΔESS); one-way ANOVA and Newman–Keuls test. A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0333.

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    Tumorigenicity of PC-3 cell subfractions: Effect of CTR. (A) Orthotopic tumors formed by PC-3v, PC-3CTRwt and PC-3CTRΔESS cells in nude mice. PC-3 cells were transfected either with vector (v), CTR-wt or CTR-ΔESS constructs. The cells (1 × 106) were orthotopically implanted into the prostate of nude mice (n = 6 per group). Animals were killed 8 weeks after implantation, and prostate tumors were weighed (A). *P < 0.01 (PC-3v vs PC-3CTRwt); ^P < 0.01 (PC3CTRΔESS vs PC-3CTRwt); one-way ANOVA and Newman–Keuls test. (B) Representative photomicrographs of H&E stained prostate sections of nude mice implanted with fast/slow adherent PC-3-CTR-wt and PC-3-CTRΔESS cells (Magnification 400×). The sections were also probed for prostate stem cell antigen immunofluorescence as described in Methods section. A full colour version of this figure is available at https://doi.org/10.1530/ERC-19-0333.

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