SHP2 is a multifunctional therapeutic target in drug resistant metastatic breast cancer

Abstract

Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of bypassing current targeted therapies via engagement of several growth promoting pathways. SH2 containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase known to facilitate growth and survival signaling downstream of numerous receptor inputs. Herein, we used inducible genetic depletion and two distinct pharmacological inhibitors to investigate the therapeutic potential of targeting SHP2 in MBC. Cells that acquired resistance to the ErbB kinase inhibitor, neratinib, displayed increased phosphorylation of SHP2 at the Y542 activation site. In addition, higher levels of SHP2 phosphorylation, but not expression, were associated with decreased survival of breast cancer patients. Pharmacological inhibition of SHP2 activity blocked ERK1/2 and AKT signaling generated from exogenous stimulation with FGF2, PDGF, and hGF and readily prevented MBC cell growth induced by these factors. SHP2 was also phosphorylated upon engagement of the extracellular matrix (ECM) via focal adhesion kinase. Consistent with the potential of SHP2-targeted compounds as therapeutic agents, the growth inhibitory property of SHP2 blockade was enhanced in ECM-rich 3D culture environments. In vivo blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis and extended the survival of systemic tumor-bearing mice. Finally, inhibition of SHP2 in combination with FGFR-targeted kinase inhibitors synergistically blocked the growth of MBC cells. Overall, our findings support the conclusion that SHP2 constitutes a shared signaling node allowing MBC cells to simultaneously engage a diversity of growth and survival pathways, including those derived from the ECM.

Fig. 1. Depletion of SHP2 inhibits the growth of metastatic breast cancer cells.

a, b Immunoblot analyses with quantification for SHP2 in 4T1 and D2.A1 cells stably expressing three independent doxycycline-inducible shRNA sequences targeting PTPN11 with and without doxycycline induction, compared with a scrambled (scram) shRNA control. c Quantification of SHP2 depletion for each shRNA construct in 4T1 and D2.A1 cells (n = 3). Depletion efficiency was defined as the percentage of SHP2 protein decrease normalized to scramble controls upon doxycycline induction. d Schematic representation of 2D and 3D growth conditions. e 2D growth assays showing differential cell viability of 4T1 and D2.A1 cells upon doxycycline-induced depletion of SHP2. Data are the mean ± s.e.m. of two biological replicates completed in triplicate. f Representative photos showing 3D morphologies of 4T1 and D2.A1 cells upon depletion of SHP2. g Quantification of differential cell viability of 4T1 and D2.A1 cells upon depletion of SHP2. Data are shown as mean ± s.e.m. (n = 3 for 4T1; *p < 0.05; n = 4 for D2.A1, **p < 0.01).

Fig. 2. Pharmacological inhibition of SHP2 blocks in vivo pulmonary metastasis.

a Schematic representation of the 4T1 post-surgical model of metastasis. b Representative bioluminescent images taken at Day 0 and Day 11 of SHP099 treatment. c Bioluminescent values of pulmonary regions of interest (ROI) were quantified as a measure of metastasis. Data are the ratio of luminescence values at Day 11 of treatment compared to Day 0. (*p < 0.05 for n = 5 mice per group). d Representative photos of pulmonary metastases (arrow heads) and H&E staining of pulmonary histological sections in control and SHP099 treated mice. e, f Plots comparing numbers of pulmonary nodules and lung weights from control and SHP099 treated mice (*p < 0.05, **p < 0.01, n = 5 mice per group). g Schematic of the D2.A1 model of pulmonary tumor growth. h Representative images of pulmonary growth monitored by bioluminescence at Day 7 and Day 21 post injection. i Bioluminescent values from pulmonary ROI quantified as the ratio of day 21 to day 7 following tumor cell injection (*p < 0.05, n = 5 mice per group). j Kaplan–Meier analyses of control and SHP099 treated mice, bearing D2.A1 pulmonary tumors, resulting in the indicated p value (n = 5 mice per group).

Fig. 3. The extracellular matrix promotes SHP2 phosphorylation.

a Cell viability of 4T1 cells cultured in 2D for 6 days in the absence or presence of the indicated concentrations of 11a-1 and SHP099. b Representative photomicrographs and quantification of 4T1 cell viability after 16 days in 3D culture in the absence or presence of the indicated concentrations of 11a-1 and SHP099. For a and b data are normalized to untreated controls and are the mean ± s.e.m. of three experiments where **p < 0.01 and ***p < 0.001. Immunoblot analyses showing differential phosphorylation of SHP2 at Y542 in 4T1 cells following 3D culture (c) or in 4T1 cells lysed directly from gel-based 3D culture (d). e Kaplan–Meier analyses of patients from the TCGA breast cancer dataset separated into two groups based on the median mRNA expression value of SHP2 (left) or median phosphorylation levels of SHP2 at Y542 (right). Overall survival was analyzed by a log-rank test resulting in the indicated p values. f RPPA data from the TCGA breast cancer dataset were analyzed for correlation of total expression levels and post translational modifications of the indicated proteins in relation to SHP2-Y542 phosphorylation. The heat map indicates the Pearson correlation coefficient and the size of the circle is representative of the value. g Immunoblot analyses showing differential phosphorylation of Src in 4T1 cells following culture under gel-based 3D conditions as compared to 2D culture. h Immunoblot analyses showing differential phosphorylation of FAK at Y925 in 4T1 cells isolated directly from gel-based 3D cultures as compared to 2D culture. i Representative photomicrographs showing 4T1 cells cultured on FN-coated tessellated scaffolds. j Immunoblot analyses showing differential phosphorylation of FAK, Src, and SHP2 in 4T1 cells cultured on FN-coated scaffolds as compared to 2D culture. k 4T1 cells were cultured on FN-coated scaffolds for 16 days and treated with the indicated concentrations of a Src inhibitor (PP2) or a FAK inhibitor (PF271) for the last 24 h before harvesting cells for immunoblot analyses of SHP2 phosphorylation at Y542. All immunoblots shown are representative of at least three independent experiments.

Fig. 4. Drug resistant breast cancer cells can be targeted by inhibition of SHP2.

a Representative dose response of HME2 parental (HME2-Par) and lapatinib resistant (HME2-LAPR) cells treated with neratinib for 6 days (right). The IC₅₀ values for each independent experiment were calculated (n = 4) and analyzed using a two-tail student t test where *p < 0.05. b Immunoblot analyzes showing differential expression of FGFR1 and phosphorylation of SHP2 at Y542 in HME2-LAPR cells compared to their HME2-Par counterparts. c Representative dose response upon 6 days treatment with 11a-1. (right) Cell viability upon treatment with 10 μM 11a-1 for each independent experiment was calculated (n = 4) and analyzed using a two-tail student t test where **p < 0.01. d Representative dose response curves of HME2 parental (HME2-Par) and HME2-LAPR cells treated with SHP099 for 6 days. (right) The IC₅₀ values for each independent experiment were calculated (n = 3) and analyzed using a two-tail student t test where **p < 0.01. e Immunoblotting showing phosphorylation of AKT, ERK1/2, and HER2 in HME2-Par and HME2-LAPR cells upon the indicated neratinib treatments. f (top) Schematic representation of the signaling recovery assays. HME2-LAPR cells were treated with neratinib for 1 h, the drug was removed, and the cells were allowed to recover in serum free media in the presence or absence of SHP2 or FGFR inhibitors for 12 h. DMSO was used as a vehicle control. (bottom) Recovery of AKT, ERK1/2, and HER2 phosphorylation were analyzed by immunoblot. g, h HME2-LAPR spheres were formed in a round bottom plate and then plated onto a bed of gel matrix in the presence of the indicated concentrations of SHP2 or FGFR inhibitors. The growth of spheroids was further induced by addition of exogenous FGF2 (20 ng/ml). The area of the sphere 9 days after placement on the ECM was measured, and these values were normalized to the initial sphere size (Day 0). Data are the mean ± s.e.m. (n = 3) were ***p < 0.001.

Fig. 5. SHP2 facilitates signaling in response to several growth factors.

a Immunoblot analyses showing the phosphorylation of SHP2 and ERK1/2 (ERK) induced by the indicated growth factors in D2.A1 cells. D2.A1 cell growth was induced by addition of exogenous FGF2, PDGF, and hGF in 2D (b) or 3D (c) culture for 6 days. D2.A1 cells expressing doxycycline inducible shRNAs targeting PTPN11 were treated with doxycycline in 2D culture (d) or 3D culture (e) in the presence of FGF2, hGF, or PDGF as indicated. For b–e cell viability was quantified as a relative bioluminescence ratio normalized to day 0. Data are the mean ± s.e.m. for at least three independent assays resulting in *p < 0.05, **p < 0.01, ***p < 0.001, or no significance (NS) as determined by a two-tail Student’s t test. f–h D2.A1 cells were pre-treated with SHP099 or FIIN4 for 24 h in serum-free media and cells were subsequently induced for 5 min with FGF2, PDGF, or hGF as indicated. Immunoblot analyses were used to detect phosphorylation of FGFR, FRS2, ERK1/2, AKT, and SHP2. i D2.A1 cells were pre-treated with PP2 or PF271 for 24 h in serum-free media, and cells were then induced for 5 min with FGF2, PDGF, or hGF. Immunoblot analyses were used to detect phosphorylation of SHP2. All immunoblots are representative of at least three independent experiments.

Fig. 6. Inhibition of SHP2 synergizes with blockade of FGFR in vitro and in vivo.

a 4T1 cells were seeded in 2D culture, and treated with the indicated concentrations of SHP099, FIIN4 alone, or both compounds for 6 days. b, c Single cell suspensions of 4T1 cells were plated in 3D matrices and treated with the indicated compounds alone or in combination for 12 days. Media containing DMSO was used as a vehicle control. Data in a and c data are bioluminescence values normalized to DMSO controls and are the mean ± s.e.m. of at least three independent experiments where **p < 0.01, ***p < 0.001 as determined by a two-tail Student’s t test. d Bioluminescence values for pulmonary regions of interest (ROI) from mice bearing 4T1 metastases normalized to values at the initiation of treatment. Data are the mean ± s.e.m. of 5 mice per treatment group. e (bottom) Representative bioluminescent images of metastasis and (top) quantified pulmonary ROI values from mice bearing 4T1 metastases 14 days after treatment initiation. Data are the mean ± s.e.m. of 5 mice per group resulting in *p < 0.05 as determined by a two-tail Mann Whitney test. f Differential survival analyses of tumor bearing mice in each treatment group, resulting in the indicated p values as determined by a log-rank test.

Fig. 7. SHP2 is a shared node for ECM and RTK signaling. Activation of FAK via integrin engagement of ECM components drives SHP2 phosphorylation.

Growth factor receptor signaling additionally contributes to phosphorylation of SHP2 via Src and FAK. SHP2 activity contributes to various downstream signaling pathways that facilitate metastatic tumor growth in the presence of currently used targeted therapies. Targeted inhibition of SHP2 (SHP2i), enhances responses to existing therapeutics. Figure was created using BioRender.