Apoptosis of estrogen-receptor negative breast cancer and colon cancer cell lines by PTP alpha and src RNAi

Abstract

We show that siRNA-mediated suppression of protein tyrosine phosphatase alpha (PTP alpha) reduces Src activity 2 to 4-fold in breast, colon and other human cancer cell lines. Src and PTP alpha RNAi induced apoptosis in estrogen receptor (ER)-negative breast cancer and colon cancer cells, but not in immortalized noncancerous breast cells, ER-positive breast cancer cells or other cancer cell types tested. RNAi of other Src family members (Fyn and Yes) or of PTP1B, a phosphatase previously suggested to be an activator of Src in breast cancer, had no effect. Although further tests with primary tumor tissues are required, the unexpected correlation between ER status and Src/PTP alpha dependence in breast cancer cell lines may be important for planning therapeutic strategies, and the insensitivity of normal breast cells to the RNAi highlights the potential of PTP alpha, which may be easier to target than Src, as a therapeutic target in ER-negative breast cancer.

Figure 1

Src and PTPα expression levels and enzymatic activities. (a) Expression levels of Src or PTPα were measured by immunoblotting cell lysates containing equal amounts of total cell protein with monoclonal antibody MAb327 or anti-PTPα polyclonal antibody 7-091 respectively. (b) Src kinase or PTPα generic phosphatase activities were measured in immune complex assays using enolase or Src-phosphorylated MBP as substrates. Relative specific activities are displayed. All values are normalized to those of MDA-MB-231. In both panels, values are normalized to those of MDA-MB-231 and error bars indicate the standard error of the mean from 3 or more independent experiments.

Figure 2

Effect of PTPα and PTP1B RNAi on Src kinase specific activity. Cells were transfected with PTPα (α), PTP1B (1B) or the reverse PTPα control (R) siRNAs and lysates were prepared after 48 hr. Protein expression was assayed by immunoblotting with anti-PTPα, -PTP1B and -Src antibodies and Src protein activity was measured in the immune complex kinase assay using enolase as substrate. (a) Examples of the effects of the siRNAs in ER⁻ breast cancer (MDA-MB-231), ER⁺ breast (MCF-7), colon (HCT-116) and prostate (PC-3) cancer cell lines. Panels (top to bottom): autoradiographs of [³²P]enolase after the Src kinase assay and immunoblots of Src, PTPα and PTP1B after the indicated siRNA treatments. (b) Src kinase specific activity was measured in experiments as shown in panel (a). Specific activity (relative to activity in the reverse PTPα siRNA-treated control cells) after suppression of PTPα or PTP1B expression by RNAi was calculated as described in Material and Methods. Standard errors from 3 to 5 (all breast cancer lines, HCT-15 and PC3) or 2 (other lines) independent experiments are indicated. Src activity was suppressed by PTPα RNAi in all lines except for HB2; the reductions were statistically significant at α = 0.05 except for HT-29. None of the PTP1B RNAi effects were statistically significant.

Figure 3

Effect of Src and PTPα RNAi on anchorage-independent growth. (a) Cells were transfected with no, reverse PTPα, PTPα or Src siRNAs as indicated, suspended in semisolid medium containing soft agarose, and monitored for colony formation. (b) Colony formation (relative to colony formation by reverse PTPα siRNA-treated cells) after suppression of Src or PTPα by RNAi was measured in experiments as shown in panel (a) and was analyzed by linear regression as described in Material and Methods. Standard errors are indicated. Three or more independent experiments were performed for all cell lines except SKBR3, HT-29, PC-3 and HeLa (Src) for which 2 experiments each were used. The values for SKBR3 and HT-29 are included for completeness but should be viewed with caution since they represent extrapolations from the observed ~50% reduction of colony formation observed at Src and PTPα suppression levels of ~50–60% to colony formation at complete suppression (see text). p-values for the null hypothesis that the RNAi had no effect on colony formation were: MDA-MB-231 (Src, <10⁻⁷; PTPα, <10⁻¹⁰), MDA-MB-435S (0.004; 0.01), MDA-MB-436 (0.04; 0.09), SKBR3 (0.15; 0.05), HCT-116 (<10⁻³; <10⁻⁹) and HT-29 (0.09; 0.16). (c) The colony-forming ratios from similar experiments using Fyn, Yes and PTP1B siRNAs are shown with the Src and PTPα RNAi results from panel (b) repeated for comparison. (d) Similar colony-forming experiments were performed in representative ER⁺ and ER⁻ breast cancer and colon cancer lines with Src, PTPα and reverse PTPα siRNAs and the additional (positive control) PTPα2, PTPα3 and PTPα4, and (negative control) Src mutant, PTPα2 mutant and PTPα3 mutant siRNAs. Average colony formation (relative to no-siRNA control transfection) and standard error (calculated without correction by linear regression) from 2 or more experiments is shown. Average Src and PTPα protein suppressions by the siRNAs are shown in Table II. (S: Src and Src mutant, α: PTPα and reverse PTPα, α2: PTPα2 and PTPα2 mutant, α3: PTPα3 and PTPα3 mutant, α4: PTPα4 siRNAs).

Figure 4

Induction of apoptosis by Src and PTPα RNAi. (a) Cells were transfected with reverse PTPα, PTPα or Src siRNAs and caspase activation was measured after 96 hr using a fluorescent caspase inhibitor binding assay. Cell nuclei or caspase-positive cells were visualized by DAPI or FITC staining, respectively. (b) The fraction of caspase activation-positive cells (after subtracting the ~1–2% of caspase-positive cells in the control siRNA-treated cells) is shown. Standard errors from 3 to 5 (MDA-MB-231, MDA-MB-435, MCF-7) or 2 (all others) experiments are indicated. The RNAi-induced increases observed with MDA-MB-231 and MDA-MB-435 were statistically significant at α = 0.05 and the increases observed with MDA-MB-436 and HCT-116 (PTPα only) were significant at α = 0.10. (c) Cells were transfected with reverse PTPα, PTPα or Src siRNAs and anti-PARP1 immunoblots were prepared after 72 hr from lysates adjusted to contain equal amounts of PARP1. Cleavage of PARP1 is an indicator of apoptosis.