α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway

Abstract

Existing anticancer strategies focused on disrupting integrin functions in tumor cells or tumor-involved endothelial cells have met limited success. An alternative strategy is to augment integrin-mediated pathways that suppress tumor progression, but how integrins can signal to restrain malignant behavior remains unclear. To address this issue, we generated an in vivo model of prostate cancer metastasis via depletion of α3β1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3β1 signals through Abl family kinases to restrain Rho GTPase activity, support Hippo pathway suppressor functions, and restrain prostate cancer migration, invasion, and anchorage-independent growth. This α3β1-Abl kinase-Hippo suppressor pathway identified α3 integrin-deficient prostate cancers as potential candidates for Hippo-targeted therapies currently under development, suggesting new strategies for targeting metastatic prostate cancer based on integrin expression. Our data also revealed paradoxical tumor suppressor functions for Abl kinases in prostate cancer that may help to explain the failure of Abl kinase inhibitor imatinib in prostate cancer clinical trials. Cancer Res; 76(22); 6577-87. ©2016 AACR.

Figure 1. Loss of α3 integrin promotes prostate cancer progression and metastasis in vivo

(A) Bioluminescence imaging (BLI) of orthotopic tumors 28 d post-implantation. Color scale indicates photons/sec/cm²/sr. (B) Tumor burden versus time. Annotation “a”: mice harboring α3-KD tumors and one mouse harboring an NT control tumor were sacrificed on day 35. Annotation “b”: the remaining mice harboring NT control tumors were sacrificed on day 46. *P=0.0004, P=0.007, and P=0.001 on days 21, 28, and 35 respectively, Holm-Sidak multiple comparison test with α=0.05. (C) Gross necropsies of mice harboring α3-KD and NT control tumors on day 35. Tumor burden of both mice was ~1 X 10⁹ photons/sec. PT, primary tumor; GI, gastrointestinal tract; Kid, kidney; Li, liver; Lu, lung. (D–E) Representative images of GFP-labeled α3-KD and NT control lung metastases. (F) Quantification of lung metastasis as μm² GFP-positive cells per high-powered field (HPF). P=0.0063, unpaired t test. (G–H) Liver colonization by GFP-labeled α3-KD and NT control cells. (I) Quantification of liver colonization as % total surface area occupied by tumor cells. P=0.0094, unpaired t test. (J–K) Kidney colonization by GFP-labeled α3-KD and NT control cells. (L) Quantification of kidney colonization as % total surface area occupied by tumor cells. P=0.0185, unpaired t test.

Figure 2. Loss of α3 integrin promotes growth under low anchorage and low growth factor conditions

Tumor cell growth (A) on a 3D collagen matrix, (B) under detached conditions, floating over a poly-HEMA coated surface, and (C) on a 2D matrix under serum growth factor deprivation is shown for 3 independent trials of each condition, *P<0.0001, #P=0.0022, unpaired t test. (D) Tumor cell growth under standard tissue culture conditions in 10% fetal bovine serum. (E) Both α3-KD and NT control cells have a similar photon flux/cell and are not significantly different, P=0.5512, linear regression analysis. Error bars, ± SEM, n=6 wells per cell type/number of cells.

Figure 3. The α3-KD cells display aberrant Rho GTPase signaling and depend on RhoA for increased growth in 3D

(A&B) RhoA and RhoC activation elicited by 5 μM LPA treatment was measured by GST-Rhotekin pulldown (*P=0.0355, unpaired t test, RhoA; *P=0.0492 unpaired t test, RhoC). Graphs show means ± SEM for 4 trials. Insets show representative blots of active RhoA and RhoC. (C) Treatment with C3-transferase drove down the 3D growth of the α3-KD cells in a dose-dependent manner. (D) LPA promoted the 3D growth of the α3-KD cells and NT control cells. (E) Stable silencing of RhoA and RhoC in the α3-KD cells. Numbers indicate intensity units measured with a LiCOR blot imager. (F) 3D growth assays comparing α3 integrin-expressing NT control cells to α3-KD cells harboring a non-targeting vector (VEC) or RhoA or RhoC shRNA constructs. Bars show mean ± SEM, n=6. Differences in growth were analyzed by ANOVA with Holm-Sidak’s multiple comparison test, ***P<0.0001, **P=0.003, ^##P=0.0041, α=0.05. (G) Stable silencing of RhoA in α3-KD cells with a third shRNA; numbers indicate intensity units measured with a LiCOR blot imager. (H) Growth of α3-KD cells harboring a non-targeting vector (VEC) or the RhoA sh3 construct under 3D or detached conditions. Bars show mean ± SEM, n=6. Differences in growth were analyzed by unpaired t test, ***P<0.0001.

Figure 4. The α3-KD cells show increased YAP and TAZ expression and depend on TAZ for increased 3D growth

(A) Immunoblot of YAP and TAZ in NT control and α3-KD cells growing under detached conditions. (B) Multiple independent experiments showed increased YAP and TAZ in α3-KD cells versus NT control cells. *P<0.0001, unpaired t test. (C) Lysates of NT control and α3-KD cells growing under detached conditions were immunoblotted for CTGF. The graph shows mean CTGF levels in 3 trials after normalization to actin. (D) Serum-starved α3-KD or NT control cells were treated with 5 μM LPA for 20 h then fixed and stained for TAZ (magenta), with nuclei counterstained with DAPI (green). Arrows indicate NT control cell nuclei with reduced or absent TAZ staining compared to adjacent cytoplasm. An identical experiment was performed for YAP (not shown). (E & F) The proportions of cells with a nuclear:cytoplasmic (N:C) ratio of TAZ or YAP greater than or less than 1.0 were determined using Image J. A larger number of 7agr;3-KD cells displayed a nuclear:cytoplasmic ratio of TAZ and YAP>1 (P<0.0001 or 0.0014 respectively, Fisher’s exact test, n ≥ 40 cells). (G) Stable knockdown of YAP and TAZ in α3-KD cells. (H) Knockdown of TAZ but not YAP inhibited growth of the α3-KD cells on 3D collagen (P<0.0001, ANOVA with Tukey’s post-test; bars show mean ± SEM, n=6). Numbers below immunoblots show intensity units measured with a LiCOR blot imager.

Figure 5. Increased 3D growth of α3-KD cells is linked to impaired Abl kinase signaling

(A) The Arg/Abl2 substrate, p190RhoGAP, was immunoprecipitated from lysates of cells growing on 3D collagen in the presence of 5 μM LPA. p190RhoGAP IPs were blotted for phosphotyrosine, p120RasGAP, or total p190RhoGAP. (B) Imatinib increased the growth of NT control cells by ~25 fold, and the growth of α3-KD cells by ~3 fold, *P<0.0001, ANOVA with Tukey’s post-test. (C) A RhoA knockdown but not a non-targeting control vector (VEC) blocked the increased 3D growth of NT control cells caused by imatinib, *P<0.0001, ANOVA with Tukey post test. (D) Knockdown of Arg/Abl2 dramatically increased 3D growth of GS689.Li cells, *P<0.0001, ANOVA with Tukey’s post test. Inset: immunoblotting of Arg in cells harboring a non-targeting control vector (VEC) or Arg sh1 or sh2 constructs. (E) Knockdown of Abl1 significantly increased 3D growth of GS689.Li cells, *P<0.0001, ANOVA with Tukey’s post test. Inset: immunoblotting of Abl in cells with non-targeting control vector (VEC) or the 3 shRNA constructs. (F) Abl kinase activator, DPH, abolished the increased 3D growth of the α3-KD cells. *P<0.0001, ANOVA with Tukey’s post test. (G) DPH had no effect on α3-KD tumor cell growth under standard tissue culture conditions.

Figure 6. α3 integrin and Abl kinase activity restrain migration and invasion

(A) Cell motility tracks for NT control and α3-KD cells migrating on collagen I monitored by time-lapse microscopy, n= 20 tracks for each cell type. (B) Migration speed for NT control and α3-KD cells on collagen I for 6 independent trials; n ≥ 50 cells of each type per experiment; P=0.0056, unpaired t test. (C) High powered fields of migrated cells in a transwell invasion assay with NT control and α3-KD cells migrating towards 5 μM LPA in the lower chamber. (D) Quantification of invasion assay showing mean ± SEM of invaded cells, n=4 fields in each of 3 wells/cell type, P<0.0001 unpaired t test. (E) Time-lapse microscopy of NT control cells and α3-KD cells before and after 5 μM imatinib addition, P<0.0001, paired t test. (F) Time-lapse microscopy of NT control and α3-KD cells before and after addition of 3 μM DPH, P<0.0001, paired t test. (G) Working model. Suppressors of invasive growth are shown in blue and promoters in red.

Figure 7. Loss of α3 integrin and increased nuclear TAZ and YAP in human prostate cancer specimens

Immunohistochemical staining of α3 integrin (A&B), TAZ (C&D), and YAP (E&F) on adjacent sections of a prostate cancer tissue microarray. B, D, and F show enlarged views of the indicated fields in A, C, and E (b, benign gland; Ca, prostate carcinoma). Arrowheads indicate nuclear TAZ and YAP staining in prostate carcinoma. (G) Mean staining scores for α3 integrin, TAZ, and YAP on a 40 case microarray of human prostate cancer and matched normal tissues. Each case was represented by 4 cores of cancer and 4 cores of normal tissue. Each point represents the average score for each patient sample, across the 4 cores. The expression of α3 integrin was reduced, while nuclear expression of TAZ and YAP was increased in tumor versus normal glands *P<0.0001, paired t test. For scoring TAZ and YAP in normal glands, only luminal cells were considered.