Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival

Abstract

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer.

Fig. 1

Notch-1 is required to repress PTEN in trastuzumab-resistant cells. a Total protein lysates from BT474TS [Trastuzumab Sensitive (Sen.)] and BT474TR [Acquired Trastuzumab Resistant (Res.)] cells underwent Western blot analysis to detect HER2, HER2 phosphorylation (PY-HER2), Akt phosphorylation (PSer473-Akt1, PThr308-Akt1), total Akt1, and β-Actin protein expression. b Total lysates from both BT474TS and BT474TR cells were subjected to Western blot analysis to detect Notch-1, PTEN, and β-Actin protein levels. BT474TR cells were transfected with scrambled control (SCBi) or Notch-1 siRNA (N-1i) for 48 h. Post transfection, total cellular Notch-1, PTEN, and β-Actin protein expression was analyzed by Western blot. c Intrinsically resistant HCC1954 cells were transfected with SCBi or Notch-1i for 48 h. Post transfection, total cellular Notch-1, PTEN, and β-Actin protein expression was analyzed by Western blot. d BT474TR cells were transfected with SCBi, N-1i, and a second Notch-1 siRNA (N-1i#2) for 48 h. Post transfection, total cellular Notch-1, PTEN, and β-Actin protein expression was analyzed by Western blot. BT474TR cells (e), or HCC1954 cells (f) were transfected with SCBi or Notch-1i for 48 h. Post transfection, relative expression of Notch-1 or PTEN mRNA was measured by RT-PCR. Asterisk (*) represents statistical significance compared to SCBi from three independent experiments using a Student’s t-test

Fig. 2

Localization and expression of PTEN in trastuzumab sensitive and resistant cells and recruitment of Notch-1 to the PTEN promoter. a Confocal immunofluorescence was performed on both BT474TS and TR cells to detect PTEN protein expression and cellular localization under conditions where BT474TR cells were transfected with a control siRNA or a Notch-1 siRNA. Scale bar = 10 μm. b BT474TR and BT474TS cells were fixed, fragmented, and chromatin was immunoprecipitated by a Notch-1 or control IgG antibody. Purified DNA from Notch-1 or IgG immunoprecipitated chromatin was used to quantify NICD1 enrichment on HEY-1 or PTEN promoters by real-time PCR using primers flanking a CSL site (maps above bar graphs) (upper graph). BT474TS cells were transfected with a vector control (pcDNA3) or NICD1-expression vector (NICD1) for 48 h. Post transfection, cells were collected, fixed, fragmented, immunoprecipitated by a C-terminal Notch-1 or control IgG antibody and NICD1 enrichment on HEY-1 or PTEN promoters was quantified by real-time PCR using HEY-1 or PTEN primers that flanked a CSL element (lower graph). Asterisk (*) represents statistical significance between sensitive and resistant (upper graph) or between pcDNA3 and NICD1 (lower graph) from three independent experiments using a Student’s t-test

Fig. 3

Notch-1-mediated inhibition of PTEN is necessary for trastuzumab-resistant bulk cell proliferation a–d. BT474TR or HCC1954 cells were transfected with control siRNA (SCBi), Notch-1 siRNA, and/or PTEN siRNA for 48 h. Post transfection, cells were seeded at 100,000 cells/well in a 6 well plate and treated daily with PBS (a, c) or 20 μg/mL trastuzumab (b, d) for 10 days. Post treatment, cells were collected and counted. Mean fold increase in live cells was calculated as the number of live cells harvested at day 10/number of live cells seeded at day 0. Asterisk (*) denotes statistical significance compared to SCBi or (**) Notch-1i as calculated using a one-way ANOVA with an overall statistical significance of p < 0.0001 from three independent experiments. e Total cellular Notch-1, PTEN, Akt, β-Actin, phosphorylation of Akt-1 at threonine308 (PThr308-Akt1) and serine473 (PSer473-Akt1) protein expression from the BT474 TR lysates in a, b post 48 h PBS (left) or trastuzumab (right) treatment was detected by Western blot. Mean values from densitometric analysis of Notch-1, PTEN, and PSer473-Akt1 normalized to β-Actin or Akt, respectively, are displayed below their respective blots, and quantitated from three independent experiments using Image J

Fig. 4

Notch-1 is required to maintain ERK1/2 signaling and bulk cell proliferation by repressing PTEN. a Total protein lysates from BT474TS (Sen.) and BT474TR (Res.) (left panel) or BT474TR cells post 48 h SCBi or Notch-1i transfection (right panel) underwent Western blot analysis to detect P-ERK1/2, ERK1/2, and β-Actin protein expression. b BT474TR cells were transfected with SCBi, Notch-1i, and/or PTENi-A siRNA for 48 h. Post transfection, 100,000 cells/well were plated in a 6 well plate and treated daily with 10 μM U0126 (left) or DMSO (right) for 10 days. Post treatment, cells were harvested and total cellular Notch-1, PTEN, P-ERK1/2, ERK1/2 and β-Actin protein expression was detected by Western blot. c Cells were counted after 10 days of treatment. Mean fold increase in live cells was calculated as the number of live cells harvested at day 10/ number of cells seeded at day 0 from triplicate wells. d BT474TR cells were transfected with SCBi or Notch-1i siRNAs and MEK1DD or pEXP expression vectors for 48 h. Total cellular Notch-1, MEK1, P-ERK1/2, ERK1/2 and β-Actin protein expressions were analyzed by Western blot. e Post transfection, cells were harvested and seeded at 100,000 cells/well of a six well plate. After 10 days, the cells were harvested and counted. Mean fold increase in live cells was calculated as the number of live cells harvested at day 10/number of cells seeded at day 0 from triplicate wells. Graphs show mean plus or minus standard deviations of three replicate assays

Fig. 5

Notch-1-mediated inhibition of PTEN is necessary for survival and self-renewal of breast cancer stem cells (BCSCs) in vitro. BT474TR (a, b) or HCC1954 (c, d) cells were transfected with SCBi, Notch-1i, and/or PTENi siRNAs for 48 h. Post transfection, cells were collected and 100,000 cells/well were seeded into PBS (upper panel) or trastuzumab (20 μg/mL) (lower panel) inoculated mammosphere forming media in a 6 well plate. Mammospheres were incubated for 10 days to form primary (1°) mammospheres then harvested, disaggregated, and 100,000 live cells from the primary mammospheres were seeded into PBS or trastuzumab inoculated mammosphere forming media. The cells were incubated for 10 days to form secondary (2°) mammospheres. Before harvesting, representative photographs of primary mammosphere were taken on day 10 at ×20 magnification. Mammospheres were harvested in PBS and mammospheres over 50 μm were counted. b, d Primary (black bars) and secondary (white bars) percent mammosphere forming efficiency (% MFE) was calculated as: [(number of mammospheres counter/well)/number of cell seeded×100]. Asterisk (*) denotes statistical significance compared to SCBi or (**) Notch-1i mean %MFE as calculated using a one-way ANOVA with an overall statistical significance of p < 0.01 from three independent experiments. Scale bar = 100 μm

Fig. 6

MEK1/2-ERK1/2 inhibition reduces trastuzumab-resistant BCSC survival. a BT474TR cells were transfected with SCBi, Notch-1i, and/or PTENi-A siRNAs for 48 h. Post transfection, cells were harvested and 100,000 live cells/well were seeded into DMSO (left) or 10 μM UO126 (right) inoculated mammosphere forming media in a 6 well plate and incubated for 10 days. On day 10, representative photographs were taken at ×20 magnification. Mammospheres were harvested in PBS and counted if they were greater than 50 μm. b Mean percent mammosphere forming efficiency (% MFE) was calculated as [(number of mammospheres counted/number of cells seeded)×100] from triplicate wells. Scale bar = 100 μm

Fig. 7

Notch-1 mediated inhibition of PTEN is required for trastuzumab-resistant breast cancer stem cell xenograft tumor formation in vivo. a BT474TR cells were transfected with SCBi, Notch-1i, and/or PTENi for 48 h. Post transfection, cells were collected and seeded in mammosphere medium at 100,000 cells/well in a 6 well plate for 10 days. After 10 days, primary mammospheres were harvested and trypsinized into single cells. In total 10,000 or 100,000 live cells in 50 μL of PBS were mixed with 50 μl of Matrigel and injected bilaterally into the mammary fat pads of female athymic nude mice. Mice were monitored for tumors weekly for 12 weeks. The Kaplan–Meier curves and log rank, Mantel–Cox test (p < 0.0001) displays percent tumor free mice injected with 10,000 cells from SCBi, Notch-1i, PTENi, and Notch-1i plus PTENi siRNA transfections. b Representative photographs of SCBi, Notch-1i and/or PTENi treated BCSC tumor formation (red circles) in nude mice. c SCBi, Notch-1i, PTENi, and Notch-1i+PTENi derived tumors excised from nude mice. d Extreme Limiting Dilution Assay results to estimate stem cell frequency for 10,000 and 100,000 cells injected bilaterally into 6–8 mice for four groups is plotted as Log fraction nonresponding on the Y-axis and Dose (# of cells injected) on the X-axis. Results from the overall test for differences in stem cell frequency between groups was calculated using Chi-Square analysis. e Schematic of Notch-1 mediated inhibition of PTEN in trastuzumab-resistant cells promotes activation of the MAPK/ERK1/2 pathway to drive cell proliferation and breast cancer stem cell survival