Notch promotes recurrence of dormant tumor cells following HER2/neu-targeted therapy - PubMed (original) (raw)

Notch promotes recurrence of dormant tumor cells following HER2/neu-targeted therapy

Daniel L Abravanel et al. J Clin Invest. 2015 Jun.

Abstract

Breast cancer mortality is principally due to recurrent tumors that arise from a reservoir of residual tumor cells that survive therapy. Remarkably, breast cancers can recur after extended periods of clinical remission, implying that at least some residual tumor cells pass through a dormant phase prior to relapse. Nevertheless, the mechanisms that contribute to breast cancer recurrence are poorly understood. Using a mouse model of recurrent mammary tumorigenesis in combination with bioinformatics analyses of breast cancer patients, we have identified a role for Notch signaling in mammary tumor dormancy and recurrence. Specifically, we found that Notch signaling is acutely upregulated in tumor cells following HER2/neu pathway inhibition, that Notch signaling remains activated in a subset of dormant residual tumor cells that persist following HER2/neu downregulation, that activation of Notch signaling accelerates tumor recurrence, and that inhibition of Notch signaling by either genetic or pharmacological approaches impairs recurrence in mice. Consistent with these findings, meta-analysis of microarray data from over 4,000 breast cancer patients revealed that elevated Notch pathway activity is independently associated with an increased rate of recurrence. Together, these results implicate Notch signaling in tumor recurrence from dormant residual tumor cells and provide evidence that dormancy is a targetable stage of breast cancer progression.

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Figures

Figure 7

Figure 7. Inhibition of Notch signaling in mice reduces the burden of dormant MRD.

(A and B) Representative images from in vivo luciferase imaging of mice bearing (A) primary tumors or minimal residual neoplastic lesions or (B) minimal residual neoplastic lesions at time points before and after treatment with vehicle or GSI. The average radiance values for each image are shown. (C and D) Box-and-whisker plots of paired longitudinal analyses of luciferase imaging in mice bearing residual lesions after treatment with (C) vehicle (n = 7) or (D) GSI (n = 6), normalized to pretreatment values. The boxes extend from the 25th–75th percentiles, with the line in the middle of each box plotted at the median. The whiskers are drawn based on the Tukey method. P values calculated by repeated-measures ANOVA.

Figure 6

Figure 6. GSI treatment blocks recurrence of dormant residual tumor cells.

(A) Schematic of MTB/TAN tumor progression and treatment paradigms. (B) Kaplan-Meier survival curves showing recurrence-free survival of MTB/TAN mice after treatment with vehicle (n = 12), 150 mg/kg (n = 13_)_ or 300 mg/kg GSI (n = 11) initiated during tumor dormancy: HR 0.37, 95% CI 0.15–0.94, P = 0.037 for 150 mg/kg; HR 0.28, 95% CI 0.10–0.78, P = 0.015 for 300 mg/kg. (C) Representative immunofluorescence analysis of NICD1 in GFP+ dormant residual tumor cells in mice, 28 days following HER2/neu downregulation and 24 hours after treatment with GSI or vehicle (×40 original magnification; n = 3). (D) Kaplan-Meier survival curves showing recurrence-free survival for mice harboring MTB/TAN orthotopic tumors expressing NICD1 or MigR1 constructs treated with GSI or vehicle initiated with doxycycline withdrawal: MigR1+GSI (n = 16_)_ vs. MigR1 + vehicle (n = 14): HR 0.23, 95% CI 0.08–0.68, P = 0.008; NICD1+GSI (n = 14) vs. NICD1 + vehicle (n = 16_)_: P = NS; NICD1 + vehicle vs. MigR1 + vehicle: HR 10.0, 95% CI 3.64–27.39, P < 0.0001; NICD1+GSI vs. MigR1+GSI: HR = 25.47, 95% CI 7.94–81.69, P < 0.0001. P values calculated by the Mantel‑Haenszel method and log-rank test for trend.

Figure 5

Figure 5. Notch signaling promotes tumor recurrence following HER2/neu downregulation.

(A) Kaplan-Meier survival curves showing recurrence-free survival for mice harboring MTB/TAN orthotopic tumors expressing NICD1 (n = 18) or MigR1 (n = 21) constructs: HR = 21.8, 95% CI 8.26–57.7; P < 0.0001 by the Mantel-Haenszel method. (B) Kaplan-Meier survival curves showing recurrence-free survival for mice harboring orthotopic tumors generated from uncultured primary MTB/TAN/Rbpjfl/fl tumor cells infected with AdGFP (n = 13) or AdCre (n = 15): HR 0.30, 95% CI 0.10–0.91; P = 0.03 by the Mantel-Haenszel method.

Figure 4

Figure 4. Notch signaling promotes colony formation following HER2/neu downregulation.

(A) Representative clonogenic survival of primary MTB/TAN tumor cells transduced with vector control (MigR1), NICD1, or dnMAML expression constructs grown in the presence or absence of doxycycline for 2 weeks. (B) Quantification of the number of colonies in A. (C) Clonogenic survival of primary MTB/TAN tumor cells transduced with dnMAML expression construct or MigR1 grown in the absence of doxycycline for 3 weeks. (D) Quantification of the number of colonies in C. Scale bar: 1 cm. Data in B and D are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by 1-way ANOVA followed by the Bonferroni multiple comparisons test; n = 3.

Figure 3

Figure 3. HER2/neu represses Notch signaling through induction of HES1 and NRARP.

(A) qRT-PCR for Nrarp or Hes1 after doxycycline withdrawal with or without re-addition of doxycycline after 48 hours. Data are representative of 3 different experiments. (B and C) qRT-PCR for (B) Hey1 or (C) Dll1 expression 48 hours after doxycycline withdrawal with and without GSI treatment in cells transduced with empty vector, NRARP (top), or HES1 (bottom) expression constructs. (D) Model for crosstalk between HER2/neu and Notch signaling. MAPK and other signaling pathways downstream of HER2/neu repress DLL1 expression through upregulation of HES1, and they repress NOTCH1 expression. They further attenuate activation of Notch targets through upregulation of NRARP. Downregulation of HER2/neu abrogates this repression, thereby allowing activation of NOTCH1 by DLL1 and upregulation of Notch targets, including HEY1 and HEY2. Data in B and C are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-way ANOVA followed by the Bonferroni multiple comparisons test; n = 3.

Figure 2

Figure 2. HER2/neu downregulation activates Notch signaling.

(A–E) Western blot and qRT-PCR analyses of Notch signaling components in HER2/neu-dependent primary tumor cells derived from MTB/TAN mice: (A and B) 48 hours after doxycycline withdrawal with and without GSI treatment. (C–E) Treatment for 4 hours (C) or 48 hours (D and E) with MEKi (PD0325901), AKTi (MK 2206), or doxycycline withdrawal. Data in B and D are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by 1-way ANOVA followed by the Bonferroni (B) or Dunnett (D) multiple comparisons tests; n = 3. Western blot results are representative of 3 different experiments.

Figure 1

Figure 1. Elevated Notch signaling is associated with decreased relapse-free survival in women with breast cancer.

Forest plot representation of meta-analysis on hazard ratios for 5-year relapse-free survival as a function of estimated NOTCH1 pathway activity for 4,463 breast cancer patients across 17 individual datasets using a Notch pathway activity signature. Names and sizes of data sets, HR (center of square), and 95% CIs (horizontal line) are shown for each dataset. Sizes of squares are proportional to weights used in meta-analysis. The overall HRs (dashed vertical lines) and associated CIs (lateral tips of diamond) are shown for the random-effects model. Solid vertical line indicates no effect. The HRs represent the change in risk over half of the full range of estimated pathway activity. The overall P value was calculated using a _z_-test on the pooled hazard ratio estimate.

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References

    1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi: 10.3322/caac.20107. - DOI - PubMed
    1. Demicheli R, Abbattista A, Miceli R, Valagussa P, Bonadonna G. Time distribution of the recurrence risk for breast cancer patients undergoing mastectomy: further support about the concept of tumor dormancy. Breast Cancer Res Treat. 1996;41(2):177–185. doi: 10.1007/BF01807163. - DOI - PubMed
    1. Saphner T, Tormey DC, Gray R. Annual hazard rates of recurrence for breast cancer after primary therapy. J Clin Oncol. 1996;14(10):2738–2746. - PubMed
    1. Karrison TG, Ferguson DJ, Meier P. Dormancy of mammary carcinoma after mastectomy. J Natl Cancer Inst. 1999;91(1):80–85. doi: 10.1093/jnci/91.1.80. - DOI - PubMed
    1. Weiss RB, et al. Natural history of more than 20 years of node-positive primary breast carcinoma treated with cyclophosphamide, methotrexate, and fluorouracil-based adjuvant chemotherapy: a study by the Cancer and Leukemia Group B. J Clin Oncol. 2003;21(9):1825–1835. doi: 10.1200/JCO.2003.09.006. - DOI - PubMed

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