Newly characterized motile sperm domain-containing protein 2 promotes human breast cancer metastasis

Abstract

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide. Breast cancer metastasis results in poor prognosis and increased mortality, but the mechanisms of breast cancer metastasis are yet to be fully resolved. Identifying distinctive proteins that regulate metastasis might be targeted to improve therapy in breast cancer. We previously described MOSPD2 as a surface membrane protein that regulates monocyte migration in vitro. In this study, we demonstrate for the first time that MOSPD2 has a major role in breast cancer cell migration and metastasis. MOSPD2 expression was highly elevated in invasive and metastatic breast cancer while it was absent or residual in normal tissue and in primary in situ tumors. In vitro experiments showed that silencing MOSPD2 in different breast cancer cell lines significantly inhibited cancer cell chemotaxis migration. Mechanistically, we found that silencing MOSPD2 profoundly abated phosphorylation events that are involved in breast tumor cell chemotaxis. In vivo, MOSPD2-silenced breast cancer cells exhibited marked impaired metastasis to the lungs. These results indicate that MOSPD2 plays a key role in the migration and metastasis of breast cancer cells and may be used to prevent the spreading of breast cancer cells and to mediate their death.

Keywords: MOSPD2; breast cancer; metastasis; migration.

Figure 1

MOSPD2 is highly prevalent in invasive breast cancer tissue. (a) Tissue arrays of multiple organ tumor and normal tissues were stained with antibody to human MOSPD2, as described in “Materials and Methods.” Representative staining of various normal and tumor organs is presented. One of two independent stained arrays is shown. (b) Breast cancer tissue array containing normal, NAT and invasive ductal carcinoma tissue was stained with anti‐MOSPD2 antibody or control antibody. Representative staining is shown. (c) Examples from breast cancer tissue array representing the abundance of MOSPD2 in NAT, primary, invasive and metastatic breast cancer tissue stained with anti‐MOSPD2 antibody. (d) Scoring summary based on staining intensity of MOSPD2 from pathology diagnosis groups shown in c. NAT, normal adjacent tissue; ID, intra‐ductal carcinoma; LC, lobular carcinoma; ILC, invasive lobular carcinoma; IDC, invasive ductal carcinoma; MIDC, metastatic invasive ductal carcinoma. **p ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

MOSPD2 promotes migration of breast cancer cells. (a) Western blot for the detection of MOSPD2 protein using anti‐MOSPD2 antibody on MDA‐231 breast cancer cells transduced with sh‐Control or sh‐MOSPD2 lentiviral particles and trans‐well migration toward EGF (200 ng/mL) of these cells, as described in “Materials and Methods.” Photos of well bottoms are shown. One of four experiments is presented. (b) Proliferation assay of sh‐Control vs. sh‐MOSPD2‐treated MDA‐231 cells. One of two experiments is shown. (c) Western blot for the detection of MOSPD2 protein expression on clones derived from MDA‐231 cells transduced with CRISPR‐Control or CRISPR‐MOSPD2 lentiviral particles targeting exon 3. (d) Trans‐well migration of clones in c. Photos of well bottoms are shown. One of three experiments is presented. (e) Enumeration of cells in d by flow cytometry. (f) Western blot for the detection of MOSPD2 on clones derived from MDA‐231 cells transduced with CRISPR‐Control or CRISPR‐MOSPD2 lentiviral particles targeting exon 9. (g) Trans‐well migration of clones in f. Photos of well bottoms are shown. One of three experiments is presented. (h) Western blots and trans‐well migration of sh‐Control compared to sh‐MOSPD2‐silenced BT‐20 cells. Photos of the lower part of the membrane are shown. One of three experiments is presented. *p < 0.05, **p ≤ 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

MOSPD2 is essential for invasion of breast cancer cells. MDA‐231 (a) or ZR‐75‐1 (c) cell lines were transduced with CRISPR‐Control or CRISPR‐MOSPD2 lentiviral particles. Control and MOSPD2‐silenced clones were tested using a 3D spheroid invasion assay for their ability to transverse the basement membrane matrix in medium containing 20% FBS and EGF 20 ng/ml for 3 days for MDA‐231 or 6 days for ZR‐75‐1 cells. (b) Proliferation assay of CRISPR‐Control vs. CRISPR‐MOSPD2‐treated MDA‐231 cells in the spheroid. One of two experiments is shown. The results shown are expressed as mean ± SD. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

MOSPD2 is essential for EGF‐induced signaling pathways in breast cancer cells. Western blots on MDA‐231 breast cancer cells transduced with CRISPR‐Control or CRISPR‐MOSPD2 lentiviral particles and activated with EGF (a, d and e) or IGF (d) for 5 min. In E, the number of tyrosine residue on EGFR is indicated. One of 2–3 experiments is shown. BT‐20 (b) and ZR‐75‐1 (c) control and MOSPD2‐silenced cells were activated with EGF for 1 and 5 min. Phosphorylation of the EGFR and downstream cues are shown. EGF 20–200 ng/mL. One of three experiments is presented.

Figure 5

MOSPD2 promotes metastasis of MDA‐231 breast cancer cells. (a,b) SCID mice were injected in the mammary fat pad with MDA‐231 breast cancer cells (5 × 10⁶) transduced with sh‐Control or sh‐MOSPD2 lentiviral particles (n = 11 and n = 8, respectively). Mice were sacrificed on day 70. The ipsilateral inguinal lymph node was excised (a), lungs were harvested for H&E staining, and the tumor area was determined (b). (c) Tumor volume comparison between mice orthotopically injected with CRISPR‐Control or CRISPR‐MOSD2‐treated MDA‐231 cells. Tumor growth was monitored externally using caliper. Tumor volume = (length × width²)/2. (d,e) SCID mice were injected into the tail vein with MDA‐231 breast cancer cells (10⁶) transduced with Control‐CRISPR or MOSPD2‐CRISPR‐isolated clone 14 (n = 11 and n = 7, respectively). Mice were sacrificed after 3 weeks and their lungs were excised for histopathologic examination. The results shown are expressed as mean ± standard error of measured metastasis size. Mean tumor area comprises the maximal lung tumor area measured for each mouse. *p ≤ 0.05, **p ≤ 0.005. [Color figure can be viewed at wileyonlinelibrary.com]