ALCAM/CD166 is a TGF-β-responsive marker and functional regulator of prostate cancer metastasis to bone

Abstract

The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β. Not only is this posttranslational modification of ALCAM a marker of prostate cancer progression, the molecule is also required for effective metastasis to bone. Biochemical analysis of prostate cancer cell lines reveals that ALCAM expression and shedding is elevated in response to TGF-β signaling. Both in vitro and in vivo shedding is mediated by ADAM17. Longitudinal analysis of circulating ALCAM in tumor-bearing mice revealed that shedding of tumor, but not host-derived ALCAM is elevated during growth of the cancer. Gene-specific knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished both skeletal dissemination and tumor growth in bone. The reduced growth of ALCAM knockdown cells corresponded to an increase in apoptosis (caspase-3) and decreased proliferation (Ki67). Together, these data demonstrate that the ALCAM is both a functional regulator as well as marker of prostate cancer progression.

Figure 1. ALCAM is overexpressed in metastatic prostate cancer and correlates with patient survival

(A) ALCAM expression levels were analyzed in publicly available dataset (GDS1439, n=19) of prostate cancer. Heat map (A) and corresponding relative expression (B) of ADAM17 (i) ALCAM (ii) N-cadherin (iii), p120 (iv) and E-cadherin (v) indicated by arrows. (C) Correlation of ALCAM expression to overall survival in a publicly available dataset (GSE10645) composed of 596 prostate cancer patients. Kaplan-Meier survival curves represent the upper and lower quartile ALCAM expression. (D) Representative images from immunohistochemical staining of ALCAM membranous and cytoplasmic expression in benign to metastatic prostate cancer. Images obtained through The Human Protein Atlas.

Figure 2. Expression and shedding of ALCAM is increased in response to TGFβ

(A)ELISA analysis of ALCAM shedding in concentrated conditioned media of PC3 cells treated with indicated exogenous cytokines. TGFβ, p<0.001 (B) Expression of ALCAM by RT-PCR fold change relative to GAPDH, glyceraldehyde 3-phosphate dehydrogenase in PC3 or LNCaP cells treated with or without 10ng/ml of TGFβ for 48 hrs (C) Western blot analysis of shed ALCAM in the conditioned media and intact ALCAM in the cell lysate in LNCaP and PC3 cells. D) Western blot detection of ALCAM and Phospho-smad2 expression in PC3 cells treated with 10ng/ml TGFβ for 48 hrs. in the presence or absence of 10μM SB431542, a TGFβ receptor tyrosine kinase inhibitor. (E) RT-PCR and F) Western blot analysis of ALCAM expression in LNCaP cells transfected with vector control or T204D, dominant active TGFβ type I receptor; *p<0.05, **p<0.01.

Figure 3. ALCAM shedding correlates with tumor burden

Circulating ALCAM levels were monitored longitudinally in mice bearing subcutaneously (S.Q.) or orthotopically implanted PC3 cells. (A) Schematic representation of in vivo strategy for S.Q. and orthotopic tumor models. B and C) Circulating levels of soluble host and tumor-derived ALCAM detectable in mice bearing subcutaneous injected PC3 cells (B) or PC3 cells orthotopically implanted into the prostate (C). Levels of ALCAM are shown as a function of time (top) or tumor burden at the time of experiment completion (bottom). Each point reflects mean of duplicate measurements ± SD. Each line and corresponding R² represents a best fit linear regression analysis.

Figure 4. Ectodomain shedding of ALCAM is mediated by ADAM17 in vitro and in vivo

(A) Western blot analysis of shed ALCAM in the conditioned media and ADAM17 in the total cell lysate of PC3 transiently transfected with either scrambled siRNA or siRNA targeting ADAM17. (B) Western blot analysis of shed ALCAM in PC3 cells treated with an ADAM17-specific inhibitor (Compound-32, BMS), broad-spectrum MMP inhibitor (GM6001), or diluent control. (C)Serum levels of tumor-derived ALCAM in 10wk old SCID mice bearing PC3-luc tumors and treated with DMSO diluent or Compound-32 for three days, p=0.0005

Figure 5. Tumor-derived ALCAM mediates TGFβ-induced migration and skeletal metastasis but not primary tumor growth in the prostate

(A) Western blot analysis of lysates from parental PC3-luc and PC3-luc shRNA ALCAM (KD1) knockdown cells. (B)Quantitative analysis of tumor cell migration in parental PC3 cells and PC3 cells with shRNA-mediated knockdown of ALCAM treated with or without 10ng/ml TGFβ. C) Whole animal luciferase imaging of mice bearing orthotopic PC3-luc parental tumors or PC3-luc ALCAM knockdown tumors 6-weeks post surgery. (D) Primary tumor weights of orthotopic PC3-luc parental tumors (n=8) and PC3-luc ALCAM knockdown tumors (n=8) (E) Representative whole-animal luciferase imaging and matching x-rays 8 weeks post-intracardiac injection of PC3-luc parental (shControl) or PC3-luc ALCAM knockdown (shALCAM, KD1). (F) Average number of bone lesions in mice from PC3-luc (1.42 ± 0.26; n=31) or PC3-luc shRNA ALCAM (KD1) knockdown cells (0.16 ± 0.07; n=25). ***p<0.0001.

Figure 6. Tumor-derived ALCAM impacts metastatic growth but not incidence after intratibial injection

(A) Representative whole animal luciferase and x-ray imaging of mice post-intratibial injection of PC3-luc parental tumors (vector, n=8), and PC3-luc ALCAM knockdown tumor cells (KD2, n=8 & KD3, n=8). (B) Bioluminescent curve of intratibial tumor development in mice bearing PC3-luc vector, PC3-luc KD2 and KD3 tumors. (Two-way ANOVA with Bonferroni post-test). (C) Tumor incidence and average lesion area in the tibias of mice bearing PC3-luc vector, PC3-luc KD2 and KD3 tumors. Data represent the mean ± SEM (n=8/group);**p<0.01; ***p<0.0001. (D) Collagen I and ALCAM immunofluorescence of tumor cells within the tibias of mice bearing PC3-luc vector or PC3-luc KD2 or KD3 tumors.

Figure 7. Tumor-derived ALCAM impacts tumor survival and proliferation in the bone microenvironment of intratibial bone tumor model

A) Representative three dimensional reconstitutions of microCT images from mice injected with PC3-luc-vector, PC3-luc-KD1 and PC3-luc-KD2 tumor cells B) Boxplots of average BV/TV (bone volume/total volume) by group for the PC3-luc-vector, PC3-luc-KD2 and PC3-luc-KD3 tumor bearing mice. Data represents quartiles with dots indicating outliers (1.5x upper or lower quartile; n=16 tibias/group) *p<0.05, **p<0.01, (One-way ANOVA p=0.0047. Post-hoc Mann-Whitney) C) A boxplot representing the lesion area calculated from end-point x-ray images from the same experiment shows dramatic decrease in the lesion area in the KD2 and KD3 tumor lesions compared to vector control (One-way ANOVA p=0.0003. Post-hoc Mann-Whitney **p<0.01, ***p<0.0001. n=8/group). Lesionareas were measured using arbitrary pixel unit. D) Representative H&E stains of osteolytic bone lesions in the hind leg of mice. Outlines indicate the osteolytic tumor lesion within the bone. E) Representative immunofluorescent staining of cleaved caspase-3 positive cells (red) in tibias of PC3-luc-vector, PC3-luc-KD2 and PC3-luc-KD3 tumor-bearing mice. F) Representative immunofluorescent staining of Ki67 proliferating cells (red) in tibias of PC3-luc-vector, PC3-luc-KD2 and PC3-luc-KD3 tumor-bearing mice. Data are mean ± SEM (n=8/group); *p<0.05, **p<0.01, ***p<0.005 (One-way ANOVA; Mann-Whitney test). G) Apoptosis in the tumor-bone microenvironment as a function of total cell number was assessed by staining for cleaved caspase-3 in PC3-luc-vector, PC3-luc-KD2 and PC3-luc-KD3 tumor bearing tibias of mice 4 weeks post-injection. H) Proliferation in the tumor-bone microenvironment as a function of total cell number was assessed by staining for Ki67 in PC3-luc-vector, PC3-luc-KD2 and PC3-luc-KD3 tumor bearing tibias of mice 4 weeks post-injection.