Protein tyrosine phosphatase µ (PTP µ or PTPRM), a negative regulator of proliferation and invasion of breast cancer cells, is associated with disease prognosis

Abstract

Background: PTPRM has been shown to exhibit homophilic binding and confer cell-cell adhesion in cells including epithelial and cancer cells. The present study investigated the expression of PTPRM in breast cancer and the biological impact of PTPRM on breast cancer cells.

Design: Expression of PTPRM protein and gene transcript was examined in a cohort of breast cancer patients. Knockdown of PTPRM in breast cancer cells was performed using a specific anti-PTPRM transgene. The impact of PTPRM knockdown on breast cancer was evaluated using in vitro cell models.

Results: A significant decrease of PTPRM transcripts was seen in poorly differentiated and moderately differentiated tumours compared with well differentiated tumours. Patients with lower expression of PTPRM had shorter survival compared with those which had a higher level of PTPRM expression. Knockdown of PTPRM increased proliferation, adhesion, invasion and migration of breast cancer cells. Furthermore, knockdown of PTPRM in MDA-MB-231 cells resulted in increased cell migration and invasion via regulation of the tyrosine phosphorylation of ERK and JNK.

Conclusions: Decreased expression of PTPRM in breast cancer is correlated with poor prognosis and inversely correlated with disease free survival. PTPRM coordinated cell migration and invasion through the regulation of tyrosine phosphorylation of ERK and JNK.

Figure 1. Expression of PTPRM in breast cancer tissues.

The PTPRM transcript level was decreased in human breast cancer compared with normal breast tissues using quantitative PCR, p = 0.36.

Figure 2. PTPRM, link to tumour grade, nodal status, TNM staging and clinical outcomes of breast cancer.

A, PTPRM transcripts were decreased in the moderately and poorly differentiated cancer cells in comparison with well-differentiated tumour cells. B, lower levels of PTPRM transcripts were seen in the advanced breast cancer. PTPRM levels were higher in tumours of early TNM stage and were decreased in the TNM2, TNM3 and TNM4. C. decreased PTPRM expression was associated with lymphatic metastasis but this was not statistically significant. D, PTPRM and Nottingham Predictive Index (NPI). NPI 1 group (NPI score<3.5; n = 59) and NPI 2 group (NPI score = 3.5–5.4; n = 35), and NPI 3 group (NPI score>5.4; n = 15) represented patients with good, moderate, and poor prognosis, respectively. E, PTPRM expression was decreased in patients with poor prognosis including local recurrence, metastasis and death from the disease. F, PTPRM expression was significantly decreased in patients who died from the disease compared with that of disease-free patients. G, Reduced PTPRM transcript levels were correlated with poorer disease free survival. The average transcript level of PTPRM in NPI 2 group was used as a threshold. *, p<0.05.

Figure 3. Knockdown of PTPRM in breast cancer cells.

A and B, knockdown of PTPRM was seen in both MDA-MB-231^ΔPTPRM (A) and MCF-7^ΔPTPRM (B) cells using RT-PCR compared with their wild-type (MDA-MB-231^WT and MCF-7^WT) and empty plasmid control (MDA-MB-231^pEF and MCF-7^pEF) cells. C and D, knockdown of PTPRM in MDA-MB-231^ΔPTPRM (C) and MCF-7^ΔPTPRM cells (D) was also verified using real-time quantitative PCR compared with pEF control cells. E and F, knockdown of PTPRM in MDA-MB-231^ΔPTPRM (E) and MCF-7^ΔPTPRM cells (F) was confirmed using western blot in comparison with pEF control. *, p<0.05.

Figure 4. The effects of PTPRM knockdown on biological functions of breast cancer cells.

A and B, Knockdown of PTPRM increased the in vitro growth of breast cancer cells. C and D, Knockdown of PTPRM promoted cell-matrix adhesion in both MDA-MB-231 and MCF-7 cells. E and F, Invasiveness of both MDA-MB-231 and MCF-7 cells were also promoted after knockdown of PTPRM. **, p<0.01 and ***, p<0.001.

Figure 5. Impact on tyrosine phosphorylation of JNK and ERK.

A, immunoprecipitation and western blot showed tyrosine phosphorylation of JNK and ERK were increased in PTPRM knockdown cells, which exhibited no effect on PLCγ phosphorylation. Relative intensity of bands from three western blots was analysed using Image J software for PLCγ (B), JNK (C), and ERK (D). *, p<0.05.

Figure 6. The knockdown of PTPRM in MDA-MB-231 cell resulted in increased cell motility via JNK and ERK pathways.

A, in vitro wounding assay showed that MDA-MB-231^ΔPTPRM cells promoted cell migration. B, incubation of MDA-MB-231^ΔPTPRM cells with PLCγ small inhibitor had no effect on cell migration using ECIS. Incubation of MDA-MB-231^ΔPTPRM cells with JNK small inhibitor (C) and ERK small inhibitor (D) diminished such effect. E, the overall changes of resistance on the fifth hour with statistical analysis. *, p<0.05 and ***, p<0.001.

Figure 7. MMP9 expression and activity in MDA-MB-231 cell.

The overall MMP9 gene expression was increased in MDA-MB-231^ΔPTPRM cells using (A) RT-PCR and (B) real-time quantitative PCR. C, gelatine zymography indicated the reduced emzyme activity of MMP9 in cells treated with ERK inhibitors. 1: pro-MMP9, 2: MMP9, and 3: MMP2. *, p<0.05.

Figure 8. Potential interacting pathways and molecules involved in the functions of PTPRM in breast cancer cells.