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. 2015 Jan;156(1):58-70.
doi: 10.1210/en.2014-1590.

Sortilin regulates progranulin action in castration-resistant prostate cancer cells

Ryuta Tanimoto  1 , Alaide Morcavallo, Mario Terracciano, Shi-Qiong Xu, Manuela Stefanello, Simone Buraschi, Kuojung G Lu, Demetrius H Bagley, Leonard G Gomella, Katia Scotlandi, Antonino Belfiore, Renato V Iozzo, Andrea Morrione
Affiliations

Sortilin regulates progranulin action in castration-resistant prostate cancer cells

Ryuta Tanimoto et al. Endocrinology. 2015 Jan.

Abstract

The growth factor progranulin is as an important regulator of transformation in several cellular systems. We have previously demonstrated that progranulin acts as an autocrine growth factor and stimulates motility, proliferation, and anchorage-independent growth of castration-resistant prostate cancer cells, supporting the hypothesis that progranulin may play a critical role in prostate cancer progression. However, the mechanisms regulating progranulin action in castration-resistant prostate cancer cells have not been characterized. Sortilin, a single-pass type I transmembrane protein of the vacuolar protein sorting 10 family, binds progranulin in neurons and negatively regulates progranulin signaling by mediating progranulin targeting for lysosomal degradation. However, whether sortilin is expressed in prostate cancer cells and plays any role in regulating progranulin action has not been established. Here, we show that sortilin is expressed at very low levels in castration-resistant PC3 and DU145 cells. Significantly, enhancing sortilin expression in PC3 and DU145 cells severely diminishes progranulin levels and inhibits motility, invasion, proliferation, and anchorage-independent growth. In addition, sortilin overexpression negatively modulates Akt (protein kinase B, PKB) stability. These results are recapitulated by depleting endogenous progranulin in PC3 and DU145 cells. On the contrary, targeting sortilin by short hairpin RNA approaches enhances progranulin levels and promotes motility, invasion, and anchorage-independent growth. We dissected the mechanisms of sortilin action and demonstrated that sortilin promotes progranulin endocytosis through a clathrin-dependent pathway, sorting into early endosomes and subsequent lysosomal degradation. Collectively, these results point out a critical role for sortilin in regulating progranulin action in castration-resistant prostate cancer cells, suggesting that sortilin loss may contribute to prostate cancer progression.

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Figures

Figure 1.
Figure 1.. Sortilin is expressed at low levels in castration-resistant cells expressing progranulin and sortilin expression levels regulates motility.
A, A total of 40 μg of protein lysates from the indicated cell lines were analyzed for sortilin expression by immunoblot with anti-sortilin monoclonal antibodies. B and C, Progranulin expression in lysates and conditioned medium (CM) of various cell lines was detected by immunoblot as described (6, 7). Immunoblot for β-actin was used as loading control. Blots are representative of 2 independent experiments. D, PC3 cells were transfected with the human Sortilin vector and vector control as described in Materials and Methods. Sortilin expression was tested by immunoblot with anti-sortilin monoclonal antibodies. E, Progranulin expression in various PC3 cell lines was analyzed by immunoblot in lysates and CMs normalized over number of cells as previously described (6, 7). Blots are representative of 2 independent experiments. Densitometric analysis is expressed as arbitrary units. F, Migration was performed as described in Materials and Methods. Data are the average of 3 independent experiments run in triplicates ± SD. ***, P < .001.
Figure 2.
Figure 2.. Sortilin overexpression in PC3 cells affects progranulin levels and downstream biological responses.
Wound healing (A), invasion (B), proliferation (C), and anchorage-independent growth (D) in soft-agar were performed as described in Materials and Methods. In soft-agar assays, only colonies of more than 150 μm were counted. Data are the average of 3 independent experiments run in triplicates ± SD. *, P < .05; **, P < .01; ***, P < .001.
Figure 3.
Figure 3.. Progranulin depletion in PC3 cells inhibits motility, invasion, proliferation, and anchorage-independent growth.
A, PC3 cells, stably depleted of endogenous progranulin, were generated by transfecting the pRS-shRNA-control (scrambled shRNA) and pRS/shPGRN plasmids. Cells were selected in medium supplemented with 2 μg/mL of puromycin. After selection, pools of progranulin-depleted PC3 cells were tested for progranulin expression levels in cell lysates and conditioned media by immunoblot using anti-progranulin polyclonal antibodies as previously described (6, 7). Blots are representative of 2 independent experiments. Densitometric analysis is expressed as arbitrary units. Migration (B), wound healing (C), invasion (D), proliferation (E), and anchorage-independent growth (F) in soft-agar were performed as described in Materials and Methods. In soft-agar assays, only colonies of more than 150 μm were counted. Data are the average of 3 independent experiments run in triplicates ± SD. ***, P < .001.
Figure 4.
Figure 4.. Sortilin depletion in PC3 cells enhances progranulin levels and downstream biological responses.
A, PC3 cells stably depleted of endogenous sortilin were generated by transfecting the pRS-shRNA-control (scrambled shRNA) or pRS/shSORT plasmids using the TransIT-Prostate Transfection kit. Cells were selected in medium supplemented with 2 μg/mL of puromycin. After selection, pools of sortilin-depleted PC3 cells were tested for sortilin expression by immunoblot using anti-sortilin monoclonal antibodies (R&D Systems). Densitometric analysis is expressed as arbitrary units. B, Progranulin expression levels in cell lysates and conditioned media as detected by immunoblot using anti-progranulin polyclonal antibodies as previously described (6, 7). Blots (A and B) are representative of 2 independent experiments. Migration (C), wound healing (D), proliferation (E), and anchorage-independent growth (F) in soft-agar were performed as described in Materials and Methods. In soft-agar assays, only colonies of more than 150 μm were counted. Data are the average of 3 independent experiments run in triplicates ± SD. **, P < .01; ***, P < .001.
Figure 5.
Figure 5.. The sortilin/progranulin complex preferentially internalizes through clathrin-dependent endocytosis.
Sortilin and progranulin colocalization with clathrin (A), caveolin-1 (B), EEA1 (C), and lysosomes (D) was assessed by confocal microscopy at the Kimmel Cancer Center Bioimaging Core Facility as described in detail in Materials and Methods. All images were analyzed using ImageJ (NIH Image) and Adobe Photoshop CS6 (Adobe Systems). Pictures are representative of at least 10 independent fields from 3 independent experiments. An average of 300 cells was examined for each condition.
Figure 6.
Figure 6.. Progranulin levels are stabilized by lysosomal inhibition.
Serum starved PC3/Control (A and B) and PC3/Sortilin (C and D) cells were incubated with Alexa Fluor 594-labeled recombinant progranulin with or without leupeptin on ice for 1 hour. After washings, cells were shifted to 37°C and imaged at the indicated time points. These images were also acquired every 12 seconds for 12 minutes using confocal microscopy and Zeiss AIM (Supplemental Movies 1 and 2). A and C, Insets are light microscopy views of the same areas of fluorescence images. B and D, The background-corrected Alexa Fluor 594 fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1. It is expressed as a function of time from experiments as in A and C, respectively. Bars are SD for n = 4.
Figure 7.
Figure 7.. Sortilin and progranulin levels modulate Akt stability.
Sortilin-overexpressing (A) and progranulin-depleted (B) cells and controls were tested by immunoblot for phospho-Akt and total Akt levels. As control, the levels of the PI3K were also assessed. Blots are representative of 2 independent experiments. C and D, Akt stability in the presence of 100μM CHX in SFM was assessed by measuring Akt levels by immunoblot at different time points of incubation in SFM (h) as described (28) in (C) sortilin-overexpressing and (D) progranulin-depleted PC3 cells. Blots are representative of 2 independent experiments. The intensities of protein bands were analyzed by densitometry using ImageJ software (NIH Image). Densitometric analysis is expressed in arbitrary units.
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