Figure 1.
TIMP-1 is up regulated in prostate cancer.
A. Serum TIMP-1 was significantly elevated in prostate cancer patients (p<0.001) as measured by sandwich ELISA (R&D Systems). n=16 for normal individuals (Ctrl) and n=140 for prostate cancer patients (Pca). B. Representative images show that TIMP-1 is elevated in prostate cancer stroma (n=6) comparing to their normal counterparts (n=6): TIMP-1 protein levels in human prostate cancer tissues (B-c and d) and normal human prostate tissues (B-a and b) were assessed by immunohistochemistry (IHC) using anti-human TIMP-1 antibody (R & D Systems). 1070624A: prostate adenocarcinoma sample from a 73 year old WM (Gleason score of 4+4=8; PSA of 5.6). 1070717A: prostate adenocarcinoma tissue from a 53 year old WM (Gleason score of 4+3=7; PSA of 8.03). Bar, 100 µm. C-D. TIMP-1 expression was elevated in more aggressive prostate cancer cell lines. TIMP-1 mRNA (C) and protein (D) expression in various prostate cancer cell lines were measured by real-time qPCR (C) and sandwich ELISA (D), respectively. TIMP-1 mRNA was normalized to beta-actin in a duplex qPCR. TIMP-1 protein in conditioned media from the indicated cell lines was measured by TIMP-1 ELISA Duoset (R&D Systems).
Figure 2.
TIMP-1 promotes in vivo growth of prostate cancers.
A. Establishment of pooled populations of PC3, 22RV1, and LAPC-4 cells expressing v5 epitope tagged TIMP-1 or transduced with the empty expression vector alone (controls). Secreted v5-tagged TIMP-1 was detected by anti-v5 mAb (Invitrogen). B. TIMP-1 promotes PC3 and 22RV1 prostate cancer growth in vivo. Weights of the tumors derived from PC3 and 22RV1 prostate cancer cells expressing TIMP-1v5 or infected with the empty expression constructs (PC3-control and 22RV1-control) were measured 5 or 10-weeks, respectively, after subcutaneous implantation of the cancer cells into Rag-2/II2rg mice (Taconic). n=6. *p<0.05. C-E. Growth rates of the subcutaneous prostate tumors derived from PC3 (C), 22RV1 (D), and LAPC-4 (E) cells expressing TIMP-1v5 or transduced with the empty expression vector (controls) as indicated in the panels. The growth rates are expressed as the mean of tumor volumes (mm3) +/- SD. Six mice were used for each type of transduced prostate cells. *p<0.05.
Figure 3.
TIMP-1 promotes accumulation of the cancer-associated fibroblasts (CAFs) and promotes prostate cancer proliferation in vivo.
The tumor sections were derived from PC3/22RV1-control cells and PC3/22RV1-TIMP-1 cells. The sections were stained with H&E (A-D) to reveal the tumor histology, with anti-alpha smooth muscle actin (anti-SMA, R&D Systems) to detect the cancer associated fibroblasts (CAFs, E-H), and with anti-Ki67 antibody (BD) to highlight the proliferating prostate cancer cells (I-L). Bar, 100 µm in A-H and 50 µm in I-L. .
Figure 4.
TIMP-1 enhances expression of Snail, MMP-2, and MMP-9 in prostate cancers.
Expression levels of several EMT markers, such as Snail (A, E, I, M), Slug (B, F, J, N), MMP-2 (C, G, K, O), and MMP-9 (D, H, L, P) were assessed on the tumor sections derived from PC3/LAPC-4 control cells (A-D and I-L, respectively) and PC3/LAPC-4-TIMP-1 cells (E-H and M-P, respectively). The results show that TIMP-1 enhances expression of Snail, MMP-2, and MMP-9 but not Slug in the prostate cancer tissues. Bar, 50 µm in A, B, E, F, I, J, M, N and 100 µm in C, D, G, H, K, L, O, P.
Figure 5.
TIMP-1 is elevated in the stroma of more aggressive human colon cancer and TIMP-1 promotes growth of human colon cancers in vivo.
A. Representative images show that TIMP-1 level is elevated in the stroma of colon cancer (n=6, top middle and bottom panels) comparing to that of normal colon (n=6, top left panel). Stroma of undifferentiated/higher grade colon cancer (n=6, the top middle panel, Z4250) and metastatic colon cancers (n=6, bottom middle and bottom right panels, Z4250 and 4081525) displayed higher levels of the stroma associated TIMP-1 comparing to that of differentiated/lower grade colon cancer (bottom left, Z4265), primary colon cancer (top middle panel), or the normal counterparts (normal colon, top left; normal liver, top right panel). Bar, 100 µm. B. Western blot analyses using anti-v5 antibody (Invitrogen) were performed to assess expression of exogenous v5-epitope tagged TIMP-1 (TIMP-1v5) in HCT116 (left panel) and HT-29 (right panel) human colon cancer cells. C-D. TIMP-1 promotes HCT116 and HT-29 colon cancer growth in vivo. Weights of the tumors derived from HCT116 and HT-29 colon cancer cells expressing TIMP-1v5 or infected with the empty expression constructs (controls) were measured 6 weeks after the subcutaneous implantation of the cancer cells. n=6. *p<0.05.
Figure 6.
TIMP-1 promotes the prostate CAF proliferation and migration through transwells.
A-C. Prostate CAFs or prostate cancer cells were seeded at 2 x 103 cells/well into 96-well plates in triplicate. Prostate cancer cell and prostate CAF proliferation assays were performed every day using a set of 96-well plates using Premix WST1 kit (TaKaRa) following the manufacturer’s instruction. *p<0.05. D. Prostate CAFs were assessed for their motility across transwell barrier over the course of 30 hours in the presence or absence of 250ng/ml of TIMP-1. 0.5 x 106 cells/ml prostate CAFs were placed in the upper chambers of Transwell inserts (Costar) in triplicates. Representative images of prostate CAF cells migrated through the transwell inserts are shown. The prostate CAFs migrated through transwell inserts in 20 random selected 200 x microscopic fields were counted. *p<0.05. A-D, the results show representative means +/-SDs of triplicates of one of two independent experiments.
Figure 7.
TIMP-1 knockdown inhibits prostate CAF proliferation and migration through transwells.
A. Relative levels of TIMP-1 in prostate CAFs infected with non-targeting shRNAs or shRNAs against TIMP-1 were assessed by ELISA and the results showed that two TIMP-1 shRNAs knocked down TIMP-1 expression by ~70% and ~50%, respectively. B. Prostate CAFs were seeded at 4 x 103 cells/well into 96-well plates in triplicate and prostate CAF proliferation assays were performed every day using a set of 96-well plates using Premix WST1 kit (TaKaRa). *p<0.05. C. Prostate CAFs were assessed for their motility across transwell barrier over the course of 30 hours in the presence or absence of 250ng/ml of TIMP-1. 1 x 106 cells/ml prostate CAFs were placed in the upper chambers of Transwell inserts (Costar) in triplicates. The prostate CAFs migrated through transwell in 20 random selected 200 x microscopic fields were counted. *p<0.05.
Figure 8.
Prostate CAFs express a higher level of TIMP-1 receptor, CD63, and TIMP-1 promotes activation of ERK1/2 kinases in prostate CAFs.
A. Expression of CD63 protein in various human prostate cancer cell lines, human prostate fibroblasts (HPrF), and human prostate CAFs (PCAFs) was determined by western blotting using anti-CD63 antibody, which recognizes heterogeneous glycosylated CD63 protein. Actin was used as a control for protein loading (lower panel). B-C. Serum-starved prostate CAF cells (B) and 22RV1 prostate cancer cells (C) were supplied with serum free medium (SFM) or SFM containing 250ng/ml of TIMP-1 for 3 and 12 hours. The cells were lysed and proteins were analyzed by Western blotting using anti-phospho-ERK1/2 or anti-phospho-AKT antibodies (upper panels), or anti-ERK1/2 or anti-AKT antibodies (bottom panels).
Figure 9.
A model of action of TIMP-1 during tumor progression
: Increased expression of TIMP-1 by tumor cells and CAFs leads to increased CAF proliferation and migration through binding of TIMP-1 to the TIMP-1 receptor, CD63 expressed on CAFs, which leads to accumulation of CAFs within cancer tissues. These CAFs in turn provide a pro-tumor microenvironment to facilitate the cancer progression by secreting pro-tumor growth factors/cytokines, modulating tumor angiogenesis, and regulating infiltration and expansion of inflammatory cells.