PDZ and LIM domain protein 1(PDLIM1)/CLP36 promotes breast cancer cell migration, invasion and metastasis through interaction with α-actinin

Abstract

Increased CLP36 expression has been found to be closely associated with breast cancer progression. However, whether and how it contributes to malignant behavior of breast cancer cells were not known. We show here that CLP36 is critical for promoting breast cancer cell migration and invasion in vitro and metastasis in vivo, whereas it is dispensable for breast cell proliferation and anchorage-independent growth in vitro and tumor growth in vivo. CLP36 interacted with both α-actinin-1 and -4 in breast cancer cells. Depletion of either α-actinin-1 or -4 inhibited breast cancer cell migration. Furthermore, mutations inhibiting the α-actinin-binding activity abolished the ability of CLP36 to promote breast cancer cell migration. Finally, depletion of CLP36 or disruption of the CLP36-α-actinin complex in breast cancer cells substantially inhibited Cdc42 activation, cell polarization and migration. Our results identify CLP36 as an important regulator of breast cancer cell migration and metastasis, and shed light on how increased CLP36 expression contributes to the progression of breast cancer.

Figure 1. CLP36 is critical for breast cancer cell migration and invasion but not proliferation and anchorage independent growth

(A) Depletion of CLP36. MDA-MB-231 cells were transfected with CLP36 siRNA KD1, KD2 or a control siRNA. The cell lysates (6 μg proteins/lane) were analyzed by Western blotting with antibodies for CLP36 or tubulin (as a loading control). (B) Haptotactic cell migration was analyzed using Transwell motility chambers in which the undersurface of the membrane was coated with fibronectin. Migration of the KD1 and KD2 cells were compared to that of the control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control. (C) Random cell migration was analyzed using Transwell motility chambers in which both surfaces of the membrane were coated with fibronectin. Migration of the KD1 and KD2 cells were compared to that of the control cells (normalized to 1). The experiment was performed twice and similar results were obtained. Panel C shows the results from a representative experiment (bars represent means ± S.D. from duplicate chambers). (D) Cell invasion was analyzed as described in the Materials and Methods. Invasion of the KD1 and KD2 cells were compared to that of the control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control. (E) Cell adhesion on fibronectin was analyzed as described in the Materials and Methods. Adhesion of the KD1 and KD2 cells were compared to those of the control cells (normalized to 1). Bars represent means ± S.D. from four independent experiments. (F) Anchorage-independent growth was analyzed as described in the Materials and Methods. The experiment was performed twice and similar results were obtained. Panel F shows the results from a representative experiment (bars represent means ± S.D. from triplicate dishes). (G) Cell proliferation was analyzed as described in the Materials and Methods. The experiment was performed twice and similar results were obtained. Panel G shows the results from a representative experiment (bars represent means ± S.D. from triplicate wells). (H) Control and CLP36 knockdown MDA-MB-231 cells were stained with a mouse anti-vinculin antibody and Rhodamine Red^™-conjugated anti-mouse IgG antibodies. Actin filaments were detected with FITC-conjugated phalloidin. Bar, 10 μm. Because depletion of CLP36 did not significantly inhibit cell-fibronectin adhesion and depletion of CLP36 resulted in similar reductions of cell migration using either haptotactic or random migration assay, the assay for haptotactic migration, which is highly relevant to cancer cell invasion and metastasis, was used for cell migration experiments shown in other figures.

Figure 2. Depletion of CLP36 diminishes breast cancer metastasis potential

(A) MDA-MB-231-Luc cells were infected with shCLP36-1, shCLP36-2 or a control lentiviral vector. The cells were analyzed by Western blotting with antibodies recognizing CLP36 or β-actin (as a loading control). (B) The mice were divided into three groups (n=9 mice per group), 6 × 10⁵ cells were injected into each mouse via tail vein. Ten weeks later, the mice were anaesthetized, and metastasis was determined using a Xenogen optical in vivo imaging system. Bioluminescence signals of a representative mouse from each group are shown. All images were obtained with the same settings (4 min exposure; photon signal: color scale from 9 × 10⁴(min) to 1.5 × 10⁵(max)). (C) Quantitative analysis of metastasis. The value of bioluminescence signals from each group were quantified and expressed as photon counts per area. Bars represent the values of each group (means ± S.D.). P values were obtained using Mann-Whitney U test. *P < 0.05 and **p < 0.01 versus the control. (D) Tumor growth. In vivo tumorigenesis was assessed as described in the Matherials and Methods. shCLP36-1, shCLP36-2 or shControl cells (6 × 10⁶ cells/mouse) were injected into the mammary fat pads of NOD/SCID mice (n=10 for each cell type). The tumors were removed and weighted twelve weeks after the mammary fat pad injection. Data represent means ±SEM.

Figure 3. CLP36 interacts with α-actinin-1 and -4 in breast cancer cells

(A and B) MDA-MB-231 cells were dually stained with mouse anti-α-actinin antibody and goat anti-CLP36 antibody. The primary mouse and goat antibodies were detected with secondary Rhodamine Red^™-conjugated anti-mouse IgG antibody and ^™2-conjugated anti-goat IgG antibody, respectively. Bar, 10 μm. The immunofluorescent images were pseudo-colored and merged using NIH Image J program (C). (D) MDA-MB-231 cell lysates were mixed with an anti-CLP36 antibody or a control antibody that does not recognize CLP36. CLP36 and control immunoprecipitates were analyzed by Western blotting with anti-CLP36 and anti-α-actinin antibodies. (E) MDA-MB-231 cell lysates were mixed with an anti-RIL antibody or a control antibody that does not recognize RIL. RIL and control immunoprecipitates were analyzed by Western blotting with anti-RIL and anti-α-actinin antibodies. (F) Lysates of MDA-MB-231 cells transfected with FLAG, FLAG-α-actinin-1 or -4 were incubated with an anti-CLP36 antibody. Anti-CLP36 immunoprecipitates were analyzed by Western blotting with anti-CLP36 and anti-FLAG antibodies. (G) Lysates of MDA-MB-231 cells transfected with FLAG, FLAG-α-actinin-1 or FLAG-α-actinin-4 construct were incubated with an anti-CLP36 antibody. Anti-CLP36 immunoprecipitates were analyzed by Western blotting with anti-CLP36 and anti-FLAG antibodies and detected with an ultra-sensitive chemiluminescent substrate (Thermo Scientific product number 34096). The samples in lanes 2 and 6 were identical to those of lanes 3 and 7 except that the cells were transfected with 25% less amount of the FLAG-α-actinin-4 construct (to ensure that the expression level of FLAG-α-actinin-4 was not higher than that of FLAG-α-actinin-1). (H) MDA-MB-231 cell lysates were mixed with an anti-CLP36 antibody or a control antibody that does not recognize CLP36. CLP36 and control immunoprecipitates were analyzed by Western blotting with anti-CLP36 and anti-α-actinin 1 antibodies. (I) α-actinin knockdown. MDA-MB-231 cells were transfected with α-actinin-1 siRNA (A1-KD1 or A1-KD2), α-actinin-4 siRNA A4-KD or a control small RNA. The cell lysates were analyzed by Western blotting with antibodies for α-actinin-1, α-actinin or tubulin (as a loading control) as indicated in the figure. (J) Haptotactic cell migration was analyzed as in Fig. 1B. Migration of the A1-KD1, A1-KD2 and A4-KD cells were compared to that of the control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control.

Figure 4. Overexpression of CLP36, but not that of RIL, promotes breast cancer cell migration

(A) FLAG-CLP36 and FLAG-RIL expression. Lysates (10 μg/lane) of MDA-MB-231 cells transfected with FLAG, FLAG-CLP36 or FLAG-RIL expression vector were analyzed by Western blotting with antibodies recognizing FLAG or tubulin (as a loading control). (B) Haptotactic cell migration was analyzed as in Fig. 1B. Migration of FLAG-CLP36 and FLAG-RIL expressing cells was compared to that of FLAG control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control. (C) α-actinin interaction. FLAG-CLP36 or FLAG-RIL was immunoprecipitated from lysates of the cells transfected with FLAG, FLAG-CLP36 or FLAG-RIL vector with M2 anti-FLAG antibody. The cell lysates (lanes 1–3) and immunoprecipitates (lanes 4–6) were analyzed by Western blotting with antibodies for α-actinin or FLAG as indicated. (D) Cell proliferation was analyzed as described in the Materials and Methods. The experiment was performed twice and similar results were obtained. Panel D shows the results from a representative experiment (bars represent means ± S.D. from triplicate wells). (E) Anchorage-independent growth was analyzed as described in the Materials and Methods. The experiment was performed twice and similar results were obtained. Panel E shows the results from a representative experiment (bars represent means ± S.D. from triplicate dishes).

Figure 5. α-actinin binding is essential for CLP36-mediated promotion of breast cancer cell migration

(A) MDA-MB-231 cells transfected with FLAG, FLAG-CLP36, FLAG-ΔZM or FLAG-ΔPDZ were analyzed by immunoprecipitation with M2 anti-FLAG-antibody. The cell lysates (lanes 1–4) and anti-FLAG immunoprecipitates (lanes 5–8) were analyzed by Western blotting with antibodies for α-actinin or FLAG as indicated. (B) Haptotactic cell migration was analyzed as in Fig. 1B. Migration of FLAG-CLP36, FLAG-ΔZM and FLAG-ΔPDZ expressing cells was compared to that of FLAG control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control.

Figure 6. Depletion of CLP36 inhibits Cdc42 activation and cell polarization

(A) Lysates of the control and CLP36 knockdown cells were analyzed by Western blotting with antibodies recognizing CLP36, Cdc42 or tubulin (as a loading control) as indicated. (B) Cdc42 activation in control and CLP36 knockdown cells were analyzed as described in the Materials and Methods. Cdc42 activation in CLP36 knockdown cells was compared to that of control cells (normalized to 1). Bar represents mean ± S.D. from three independent experiments. *P < 0.05. (C) Lysates of the control and CLP36 knockdown cells were analyzed by Western blotting with antibodies recognizing CLP36, phospho-Akt (Ser473), Akt, phospho-Erk1/2 (Thr202/Tyr204), Erk1/2, Cdc42 or tubulin (as a loading control) as indicated. (D and E) Cell polarity. MDA-MB-231 cells transfected with control RNA or CLP36-specific siRNA were subjected to scratch wounding. The cells were stained for Golgi (GM130; red) and nuclei (DAPI; blue) (D). Bar, 10 μm. Cell polarity was analyzed as described in the Materials and Methods (E). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control.

Figure 7. Dominant negative inhibition of the CLP36-α-actinin complex

(A) FLAG-ΔLIM expression and its binding to α-actinin. MDA-MB-231 cells were infected with adenoviral vectors encoding FLAG-ΔLIM or β-galactosidase (as a control). The cell lysates (lanes 1 and 2) and anti-FLAG immunoprecipitates (lanes 3 and 4) were analyzed by Western blotting with anti-FLAG or anti-α-actinin antibodies. (B) Inhibition of the CLP36-α-actinin complex. The cell lysates (lanes 1 and 2) and CLP36 immunoprecipitates (lanes 3 and 4) were analyzed by Western blotting with anti-α-actinin or anti-CLP36 antibodies. (C) The total lysates, Triton X-100 soluble and insoluble fractions of cells infected with adenoviral vectors encoding FLAG-ΔLIM or β-galactosidase were prepared as described in the Materials and Methods. The samples were analyzed by Western blotting with anti-α-actinin, anti-CLP36 or anti-FLAG antibodies and Coomassie blue staining as indicated.

Figure 8. Disruption of the CLP36-α-actinin complex compromises Cdc42 activation, cell polarization, migration and invasion

(A and B) Cell migration and invasion. Haptotactic cell migration (A) and cell invasion (B) were analyzed as in Figs. 1B and 1D. Migration and invasion of FLAG-ΔLIM expressing cells were compared to those of β-gal control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control. (C and D) Cell polarity. MDA-MB-231 cells infected with adenoviral vectors encoding FLAG-ΔLIM or β-galactosidase (as a control) were subjected to a scratch wounding. The cells were co-stained for Golgi (GM130; red) and nuclei (DAPI; blue) (C). Bar, 10 μm. Cell polarity was analyzed as described in the Materials and Methods (D). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control. (E and F) Cdc42 activation. Lysates of Cdc42 MDA-MB-231 cells infected with adenoviral vectors encoding FLAG-ΔLIM or β-galactosidase (as a control) were analyzed by Western blotting with antibodies for Cdc42 and tubulin (as a loading control) (E). Active Cdc42 was quantified as described in the Materials and Methods (F). Cdc42 activation in FLAG-ΔLIM expressing cells was compared to that of control cells (normalized to 1). Bar represents mean ± S.D. from three independent experiments. *P < 0.05. (G) Cell proliferation was analyzed as described in the Materials and Methods. The experiment was performed twice and similar results were obtained. Panel G shows the results from a representative experiment (bars represent means ± S.D. from triplicate wells). (H) Anchorage-independent growth was analyzed as described in the Materials and Methods. The experiment was performed twice and similar results were obtained. Panel H shows the results from a representative experiment (bars represent means ± S.D. from triplicate dishes).

Figure 9. Depletion of CLP36 inhibits BT549 breast cancer cell migration and invasion

(A) Lysates of BT549 cells transfected with FLAG, FLAG-α-actinin-1 or -4 were incubated with an anti-CLP36 antibody. Anti-CLP36 immunoprecipitates were analyzed by Western blotting with anti-CLP36 and anti-FLAG antibodies. (B) Depletion of CLP36. BT549 cells were transfected with CLP36 siRNA KD1, KD2 or a control siRNA. The cell lysates (6 μg proteins/lane) were analyzed by Western blotting with antibodies for CLP36 or tubulin (as a loading control). (C and D) Haptotactic cell migration (C) and cell invasion (D) were analyzed as in Figs. 1B and 1D. Migration and invasion of the KD1 and KD2 cells were compared to those of the control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control.

Figure 10. Disruption of the CLP36-α-actinin complex inhibits BT549 breast cancer cell migration and invasion

(A) FLAG-ΔLIM expression. BT549 cells were infected with adenoviral vectors encoding FLAG-ΔLIM or β-galactosidase (as a control) as described in the Materials and Methods. Cell lysates (lanes 1 and 2) were analyzed by Western blotting with anti-FLAG antibody and Coomassie blue staining as indicated. (B) Inhibition of the CLP36-α-actinin complex. The cell lysates (lanes 1 and 2) and CLP36 immunoprecipitates (lanes 3 and 4) were analyzed by Western blotting with anti-α-actinin or anti-CLP36 antibodies. (C and D) Cell migration and invasion. Haptotactic cell migration (C) and cell invasion (D) of FLAG-ΔLIM expressing cells were analyzed as in Figs. 1B and 1D and were compared to those of β-gal control cells (normalized to 1). Bars represent means ± S.D. from three independent experiments. *P < 0.05 versus the control.