Project description:Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable, whereas active phosphoinositide-3 kinase (PI3K)-Akt and mitogen-activated protein kinase (MAPK) were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFKs) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.
Project description:HER2 (ERBB2) gene amplification and PIK3CA mutations often co-occur in breast cancer, and aberrant activation of the PI3K pathway has been implicated in resistance to HER2-directed therapies. We have created a mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in their mammary glands developed tumors with a significantly shorter latency compared to mice expressing either oncogene alone. By microarray analysis, HER2-driven tumors clustered with the luminal subtype, whereas HER2+PIK3CA and PIK3CA-driven tumors were associated with the claudin-low breast cancer subtype. In accordance, PIK3CA and HER2+PIK3CA tumors expressed elevated levels of EMT and stem cell markers, and cells from HER2+PIK3CA tumors more efficiently formed mammospheres, providing further evidence that activated PIK3CA may enrich for cancer stem cells. Finally, HER2+PIK3CA tumors are resistant to the HER2 antibody trastuzumab; resistance is partially reversed by the addition of a PI3K inhibitor. Taken together, these studies suggest that the co-expression of HER2 and PI3KH1047R in the mouse mammary gland accelerates the formation of aggressive, trastuzumab-resistant tumors. referenceXsample
Project description:The MET receptor tyrosine kinase represents a promising target in cancer. PIK3CA activating mutations are common in several tumor types and can potentially confer resistance to anti-receptor tyrosine kinase therapy.MET and/or PI3K pathway inhibition was assessed in NIH3T3 cells harboring MET-activating point mutation with or without ectopic expression of PIK3CAE545K and PIK3CAH1047R, as well as in MET-expressing head and neck cancer cells with endogenous PIK3CA mutations. Endpoints included PI3K pathway activation, cell proliferation, colony-forming ability, cell death, wound-healing, and an in vivo model.PIK3CAE545K and PIK3CAH1047R confer resistance to MET inhibition in MET-driven models. PIK3CAH1047R was more potent than PIK3CAE545K at inducing resistance in PI3K pathway activation, cell proliferation, colony-forming ability, induction of cell death and wound-healing upon MET inhibition. Resistance to MET inhibition could be synergistically overcome by co-targeting PI3K. Furthermore, combined MET/PI3K inhibition led to enhanced anti-tumor activity in vivo in tumors harboring PIK3CAH1047R. In head and neck cancer cells the combination of MET/PI3K inhibitors led to more-than-additive effects.PIK3CA mutations can lead to resistance to MET inhibition, supporting future clinical evaluation of combinations of PI3K and MET inhibitors in common scenarios of malignant neoplasms featuring aberrant MET expression and PIK3CA mutations.
Project description:<h4>Introduction</h4>The overexpression of human epidermal growth factor receptor (HER)-2 in 20% of human breast cancers and its association with aggressive growth has led to widespread use of HER2-targeted therapies, such as trastuzumab (T) and lapatinib (L). Despite the success of these drugs, their efficacy is limited in patients whose tumors demonstrate de novo or acquired resistance to treatment. The ?1 integrin resides on the membrane of the breast cancer cell, activating several elements of breast tumor progression including proliferation and survival.<h4>Methods</h4>We developed a panel of HER2-overexpressing cell lines resistant to L, T, and the potent LT combination through long-term exposure and validated these models in 3D culture. Parental and L/T/LT-resistant cells were subject to HER2 and ?1 integrin inhibitors in 3D and monitored for 12 days, followed by quantification of colony number. Parallel experiments were conducted where cells were either stained for Ki-67 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or harvested for protein and analyzed by immunoblot. Results were subjected to statistical testing using analysis of variance and linear contrasts, followed by adjustment with the Sidak method.<h4>Results</h4>Using multiple cell lines including BT474 and HCC1954, we reveal that in L and LT resistance, where phosphorylation of EGFR/HER1, HER2, and HER3 are strongly inhibited, kinases downstream of ?1 integrin--including focal adhesion kinase (FAK) and Src--are up-regulated. Blockade of ?1 by the antibody AIIB2 abrogates this up-regulation and functionally achieves significant growth inhibition of L and LT resistant cells in 3D, without dramatically affecting the parental cells. SiRNA against ?1 as well as pharmacologic inhibition of FAK achieve the same growth inhibitory effect. In contrast, trastuzumab-resistant cells, which retain high levels of phosphorylated EGFR/HER1, HER2, and HER3, are only modestly growth-inhibited by AIIB2.<h4>Conclusions</h4>Our data suggest that HER2 activity, which is suppressed in resistance involving L but not T alone, dictates whether ?1 mediates an alternative pathway driving resistance. Our findings justify clinical studies investigating the inhibition of ?1 or its downstream signaling moieties as strategies to overcome acquired L and LT resistance.
Project description:The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.
Project description:Despite multiple advances in the treatment of HER2+ breast cancers, resistance develops even to combinations of HER2 targeting agents. Inhibition of PI3K pathway signaling is critical for the efficacy of HER2 inhibitors. Activating mutations in PIK3CA can overlap with HER2 amplification and have been shown to confer resistance to HER2 inhibitors in preclinical studies.Lapatinib-resistant cells were profiled for mutations in the PI3K pathway with the SNaPshot assay. Hotspot PIK3CA mutations were retrovirally transduced into HER2-amplified cells. The impact of PIK3CA mutations on the effect of HER2 and PI3K inhibitors was assayed by immunoblot, proliferation and apoptosis assays. Uncoupling of PI3K signaling from HER2 was investigated by ELISA for phosphoproteins in the HER2-PI3K signaling cascade. The combination of HER2 inhibitors with PI3K inhibition was studied in HER2-amplified xenograft models with wild-type or mutant PIK3CA.Here we describe the acquisition of a hotspot PIK3CA mutation in cells selected for resistance to the HER2 tyrosine kinase inhibitor lapatinib. We also show that the gain of function conferred by these PIK3CA mutations partially uncouples PI3K signaling from the HER2 receptor upstream. Drug resistance conferred by this uncoupling was overcome by blockade of PI3K with the pan-p110 inhibitor BKM120. In mice bearing HER2-amplified wild-type PIK3CA xenografts, dual HER2 targeting with trastuzumab and lapatinib resulted in tumor regression. The addition of a PI3K inhibitor further improved tumor regression and decreased tumor relapse after discontinuation of treatment. In a PIK3CA-mutant HER2+ xenograft, PI3K inhibition with BKM120 in combination with lapatinib and trastuzumab was required to achieve tumor regression.These results suggest that the combination of PI3K inhibition with dual HER2 blockade is necessary to circumvent the resistance to HER2 inhibitors conferred by PIK3CA mutation and also provides benefit to HER2+ tumors with wild-type PIK3CA tumors.
Project description:<h4>Purpose</h4>c-Src is an important adapter protein with oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), which validates it as an attractive target for the treatment of breast cancer. A specific c-Src inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo[3,4-d]pyrinidine (PP2), was utilised to block c-Src activity to identify targeted vulnerabilities affected by ER and HER2 in a panel of breast cancer cell lines.<h4>Methods</h4>ER, growth factor receptors and signalling pathways were detected by Western-blot. The DNA content of the cells was determined by using a DNA fluorescence quantitation kit. Cell cycles were analysed by flow cytometry.<h4>Results</h4>The antiproliferative effect of PP2 closely correlated with the inhibition of c-Src mediated extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt growth pathways. Inhibition of c-Src tyrosine kinase predominantly blocked ER negative breast cancer cell growth, particularly the triple (i.e. ER, progesterone receptor (PR), and HER2) negative cells. In contrast, ER negative Sk-Br-3 cells with highest HER2 phosphorylation were resistant to PP2, in which hyper-activated HER2 directly regulated growth pathways. However, blocking c-Src recovered ER expression and down-regulated HER2 which made Sk-Br-3 cells regain responsiveness to 4-hydroxytamoxifen. The majority of ER positive cells were not sensitive to PP2 regardless of wild-type or endocrine resistant cell lines.<h4>Conclusions</h4>c-Src mediates the essential role of growth pathways in ER negative breast cancer cells. The ER positive and HER2 over-activation are two important predictive biomarkers for the resistance to a c-Src inhibitor. These data provided an important therapeutic rationale for patient selection in clinical trials with c-Src inhibitors in breast cancer.
Project description:Human epidermal growth factor receptor 2 (HER2; ERBB2) amplification and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations often co-occur in breast cancer. Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to correlate with a diminished response to HER2-directed therapies. We generated a mouse model of HER2-overexpressing (HER2(+)), PIK3CA(H1047R)-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared with mice expressing either oncogene alone. HER2 and mutant PIK3CA also cooperated to promote lung metastases. By microarray analysis, HER2-driven tumors clustered with luminal breast cancers, whereas mutant PIK3CA tumors were associated with claudin-low breast cancers. PIK3CA and HER2(+)/PIK3CA tumors expressed elevated transcripts encoding markers of epithelial-to-mesenchymal transition and stem cells. Cells from HER2(+)/PIK3CA tumors more efficiently formed mammospheres and lung metastases. Finally, HER2(+)/PIK3CA tumors were resistant to trastuzumab alone and in combination with lapatinib or pertuzumab. Both drug resistance and enhanced mammosphere formation were reversed by treatment with a PI3K inhibitor. In sum, PIK3CA(H1047R) accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers, and generates resistance to approved combinations of anti-HER2 therapies.
Project description:BACKGROUND: Src is a non-receptor tyrosine kinase involved in signalling and crosstalk between growth-promoting pathways. We aim to investigate the relationship of active Src in response to trastuzumab of HER2-positive breast carcinomas. METHODS: We selected 278 HER2-positive breast cancer patients with (n=154) and without (n=124) trastuzumab treatment. We performed immunohistochemistry on paraffin-embedded tissue microarrays of active Src and several proteins involved in the PI3K/Akt/mTOR pathway, PIK3CA mutational analysis and in vitro studies (SKBR3 and BT474 cancer cells). The results were correlated with clinicopathological factors and patients' outcome. RESULTS: Increased pSrc-Y416 was demonstrated in trastuzumab-resistant cells and in 37.8% of tumours that correlated positively with tumour size, necrosis, mitosis, metastasis to the central nervous system, p53 overexpression and MAPK activation but inversely with EGFR and p27. Univariate analyses showed an association of increased active Src with shorter survival in patients at early stage with HER2/hormone receptor-negative tumours treated with trastuzumab. CONCLUSIONS: Src activation participates in trastuzumab mechanisms of resistance and indicates poor prognosis, mainly in HER2/hormone receptor-negative breast cancer. Therefore, blocking this axis may be beneficial in those patients.
Project description:INTRODUCTION: Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib. METHODS: To determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses. RESULTS: Src activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling. CONCLUSIONS: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.