Kelly L. Mueller

Met and c-Src Cooperate to Compensate for Loss of Epidermal Growth Factor Receptor Kinase Activity in Breast Cancer Cells

Breast cancers are not responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), although 30% of breast cancers overexpress EGFR. The mechanism of intrinsic resistance to EGFR TKIs in breast cancer is the focus of current studies. Here, we observed that EGFR remains tyrosine phosphorylated in breast cancer cells that proliferate in the presence of EGFR TKIs. In one such cell line, SUM229, inhibiting c-Src kinase activity with either a dominant-negative c-Src or a c-Src TKI decreased EGFR phosphorylation on Tyr(845), Tyr(992), and Tyr(1086) in the presence of EGFR TKIs. Conversely, overexpressing wild-type (wt) c-Src in the EGFR TKI-sensitive breast cancer cell line SUM149 increased EGFR kinase-independent EGFR tyrosine phosphorylation. In addition, in the presence of EGFR TKIs, inhibiting c-Src kinase activity decreased cell growth in SUM229 cells, and overexpressing wt-c-Src increased cell growth in SUM149 cells. We identified the receptor tyrosine kinase Met to be responsible for activating c-Src in SUM229 cells. Inhibiting Met kinase activity with a small molecule inhibitor decreased c-Src phosphorylation and kinase activation. In addition, inhibiting Met kinase activity in SUM229 cells decreased EGFR tyrosine phosphorylation and growth in the presence of EGFR TKIs. Stimulating Met kinase activity in SUM149 cells with hepatocyte growth factor increased EGFR tyrosine phosphorylation and cell growth in the presence of EGFR TKIs. These data suggest a Met/c-Src-mediated signaling pathway as a mediator of EGFR tyrosine phosphorylation and cell growth in the presence of EGFR TKIs.

Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib

Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including ∼30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI‐resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitors and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt‐ and mitogen‐activated protein kinase in an EGFR TKI‐sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI‐resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI‐induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signaling in the absence of EGFR kinase activity. J. Cell. Physiol. 226: 2316–2328, 2011.

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met

IntroductionEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC).MethodsThe SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Students t test was used for all statistical analysis.ResultsHere we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI.ConclusionsTaken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.

EGFR/Met association regulates EGFR TKI resistance in breast cancer

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.

Dasatinib is synergistic with cetuximab and cisplatin in triple-negative breast cancer cells.

BACKGROUND Patients presenting with triple-negative breast cancers (TNBCs) have a poorer prognosis compared with those with other subtypes of breast cancer. The majority of TNBCs overexpress epidermal growth factor receptor (EGFR). However, EGFR inhibition as a monotherapy, as with the monoclonal antibody cetuximab, is ineffective. Src family tyrosine kinases play a critical role in signal transduction downstream of growth factor receptors and are involved in the development of EGFR inhibitor resistance. We hypothesize that dasatinib, an Src family tyrosine kinase inhibitor, may help overcome EGFR resistance to cetuximab, and in combination with cisplatin may enhance growth inhibition and apoptosis and reduce metastatic potential. METHODS Growth inhibition, apoptosis, cell migration and invasion, and effects on EGFR, Akt, and mitogen-activated protein kinase phosphorylation were examined in a panel of breast cancer cell lines, including seven TNBC cell lines. RESULTS Six out of seven TNBC cell lines demonstrated a synergistic interaction using the triple-drug combination, compared with only two TNBC cell lines with the cisplatin and cetuximab combination. An induction of apoptosis and decrease in EGFR and mitogen-activated protein kinase phosphorylation, and thus resensitization to EGFR inhibition, was observed using the three-drug treatment regimen. A significant reduction (P < 0.001) in tumor cell migration and invasion was also found following dasatinib treatment alone or in combination. CONCLUSIONS These findings may have important clinical implications in treating TNBC patients whose tumors co-overexpress both EGFR and c-Src. Identification of this subset of patients may be beneficial in the design of a clinical trial using this treatment regimen.

A targeted enzyme approach to sensitization of tyrosine kinase inhibitor-resistant breast cancer cells

Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of potential use in patients with breast cancer. Unfortunately, in clinical studies, gefitinib is often ineffective indicating that resistance to EGFR inhibitors may be a common occurrence in cancer of the breast. EGFR has been shown to be overexpressed in breast cancer, and in particular remains hyperphosphorylated in cell lines such as MDA-MB-468 that are resistant to EGFR inhibitors. Here, we investigate the cause of this sustained phosphorylation and the molecular basis for the ineffectiveness of gefitinib. We show that reactive oxygen species (ROS), known to damage cellular macromolecules and to modulate signaling cascades in a variety of human diseases including cancers, appear to play a critical role in mediating EGFR TKI-resistance. Furthermore, elimination of these ROS through use of a cell-penetrating catalase derivative sensitizes the cells to gefitinib. These results suggest a new approach for the treatment of TKI-resistant breast cancer patients specifically, the targeting of ROS and attendant downstream oxidative stress and their effects on signaling cascades.

Protein Expression of DNA Damage Repair Proteins Dictates Response to Topoisomerase and PARP Inhibitors in Triple-Negative Breast Cancer

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

Constitutive c-Met phosphorylation mediates growth in the absence of EGFR kinase activity in breast cancer cells dependent on EGFR expression for growth.

Abstract #4059 Breast cancers are not responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) even though 30% of breast cancers overexpress the EGFR. We have observed that EGFR remains tyrosine phosphorylated in breast cancer cells that do not require EGFR kinase activity for growth, yet express high levels of EGFR. In one such cell line, SUM229, we found that this EGFR kinase-independent EGFR tyrosine phosphorylation encompasses all of the tyrosine phosphorylation sites of the EGFR for which antibodies are available, with the exception of tyrosine 1148. Using shRNA lentiviral constructs targeting EGFR, we also found that EGFR expression is required for the growth of the SUM229 cells, even though the kinase activity is not required. In addition, we found that this aberrant tyrosine phosphorylation and growth in the absence of EGFR kinase activity is mediated in part by c-Src and c-Met kinase activity. Interestingly, c-Met is constitutively phosphorylated in the SUM229 cells. This study describes the mechanism of c-Met constitutive activity and defines the Met-mediated signaling pathways to growth and survival in the absence of EGFR kinase activity. Specifically, through sequence analysis of mRNA from SUM229 cells, c-Met is not mutated, confirming the lack of identification of c-Met mutations in breast cancer. In addition, using publically available databases and our own immunoblotting experiments, c-Met is neither amplified nor overexpressed at the mRNA or protein level in the SUM229 cells. Similarly, ligand-mediated regulators of c-Met phosphorylation, including HGF, HGFAC, HAI-1, and HA1-2 are not overexpressed at the mRNA level and HGF protein expression and secretion are comparable to levels seen in breast cancer cells without c-Met constitutive phosphorylation. However, c-Met localization to the cell surface appears to be elevated in the SUM229 cells and c-Met co-immunoprecipitates with EGFR under these conditions. Taken together, these data suggest that c-Met and EGFR association at the cell surface may be mediating c-Met constitutive phosphorylation in the absence of EGFR kinase activity. With respect to the signaling pathways that mediate EGFR kinase-independent, yet c-Met kinase-dependent growth in SUM229 cells we have found that the phosphorylation of Akt, MAPK, p38MAPK, and JNK1/2 are unaffected by inhibition of both EGFR and c-Met kinase activities. Interestingly, the phosphorylation of Jak2, PLCg, and Gab1 are decreased when EGFR and c-Met kinases are inhibited. These data suggest that pathways signaling through Jak2, PLCg, and/or Gab1 may be mediating growth in the absence of EGFR kinase activity. In preliminary data, we know that these pathways do not involve PKC, Akt, or PI3-Kinase. Therefore, current investigations are focused on the STAT family of proteins as well as the Rho/Rac/Cdc42 mediated signaling pathways. Taken together, these data suggest that c-Met mediates a unique signaling pathway in SUM229 cells that stimulates both growth and survival in the absence of EGFR kinase activity. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4059.

Abstract 1026: Combating resistance to EGFR inhibitors: eIF4B as a novel target.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Triple negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive cancer that currently has no efficacious therapy. These patients have a high rate of relapse and often develop resistance to chemotherapy. Many TNBCs, both in vitro and in vivo, have elevated levels of epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as a monotherapy. The TNBC cell line, BT20, has increased levels of EGFR and is resistant to EGFR inhibitors. To identify the signaling pathways that remain phosphorylated after treatment with gefitinib, an EGFR tyrosine kinase inhibitor (TKI), we used mass spectrometry based phospho-proteomics. Through these assays we identified many components of the mTOR pathway phosphorylated in the presence of gefitinib and further sought to explore this pathway as a mechanism of resistance to EGFR inhibitors in TNBC. mTOR inhibitors have been investigated in the clinic due to their ability to inhibit the PI3Kinase/Akt pathway thus decreasing cell survival and proliferation. Inhibiting mTOR also importantly inhibits translation. Despite activation of these pathways our TNBC cell lines, BT20, MDA-MB-123, and MDA-MB-468 are resistant to temsirolimus, an mTOR inhibitor. Our studies have found that dual treatment with an mTOR inhibitor and an EGFR TKI has a synergistic effect on decreasing TNBC cell viability, but the mechanism of this synergy is not understood. We have found that the abrogation of both EGFR and mTOR signaling does not alter the phosphorylation status of key signaling pathways including MAPK and Akt. Instead, our preliminary data have identified the translational control protein eIF4B as potentially key fragile point in EGFR and mTOR inhibitor synergy. Therefore, in this study we hypothesized that mTOR inhibition will sensitize TNBC cells to EGFR TKIs through the inhibition of eIF4B phosphorylation. Small molecules have yet to be identified to abrogate the function of eIF4B, which when phosphorylated enhances the helicase activity of eIF4A critical to translation. Therefore, we knockdown eIF4B expression and found a decrease in cell survival comparable to the decrease observed with gefitinib and temsirolimus treatment. In addition, we have identified p70S6K and p90RSK as kinases directly responsible for eIF4B phosphorylation, such that both molecules need to be inactivated in order for eIF4B phosphorylation to be inhibited. This inactivation correlates with a loss of cell growth and viability and a decrease in clonogenic cell survival. Lastly, we have shown that the downstream functions of eIF4B phosphorylation are abrogated with EGFR and mTOR inhibitors. Therefore, taken together these data suggest that in the presence of activated MAPK and AKT, EGFR and mTOR inhibitors have the ability to abrogate cell growth, viability, and survival via disruption of the translational control mechanisms through eIF4B. Citation Format: Julie Madden, Kelly Mueller, Aliccia Bollig-Fischer, Paul Stemmer, Julie Boerner. Combating resistance to EGFR inhibitors: eIF4B as a novel target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1026. doi:10.1158/1538-7445.AM2013-1026

Abstract 1068: Inhibition of the phosphorylation of eIF4 molecules sensitizes EGFR expressing breast cancers to EGFR inhibitors

Triple-negative breast cancers have a poor prognosis and lack an effective targeted therapeutic. Even though ∼50% of triple-negative breast cancers express epidermal growth factor receptor (EGFR), they are resistant to EGFR tyrosine kinase inhibitors (TKI). Although the activation of several tyrosine kinases including Met, c-Src, and IGF-IR have been found to correlate with EGFR TKI resistance, the mechanism of this de novo resistance is still being elucidated. The BT20 breast cancer cell line has amplified EGFR expression yet is resistant to EGFR TKIs. Phospho-proteomic analysis by mass spectrometry was used to identify proteins that remain phosphorylated after gefitinib treatment. These proteins included Raptor, PDK1, and GSK3, implicating the Akt and mTOR pathways in EGFR TKI resistance. Despite activation of these pathways, BT20 cells were resistance to the mTOR antagonist temsirolimus. However, temsirolimus was able to sensitize BT20 cells to gefitinib which led to a synergistic decrease in cell survival. Similar results have been observed by others using different inhibitors; however, the mechanism of this synergistic decrease in cell survival has not been identified. These data suggest that EGFR and mTOR activation regulate cell survival through parallel signaling pathways and that both pathways need to be inhibited to abrogate growth and survival. This is supported by the observation that the PI3Kinase/Akt signaling pathway was unaffected by gefitinib or temsirolimus treatment alone. In addition, the combination of EGFR and mTOR inhibitors had no effect on PI3Kinase/Akt or Ras/MAPK signaling, despite the synergistic decrease in cell survival. However, further immunoblotting showed a pronounced inhibition in activation of the eIF4 family in the mTOR translational control pathway with dual treatment of gefitinib and temsirolimus, an abrogation not significantly observed with either treatment alone. These results suggest that inhibiting mTOR signaling alone is insufficient to modulate the translational control pathway in BT20 cells and that EGFR signals independent of mTOR to activate this pathway. These data demonstrate that phosphorylation and subsequent regulation of these translational control molecules may be critical to the survival of BT20 cells. Indeed, the combination of gefitinib and temsirolimus stimulated apoptosis in BT20 cells, supporting the idea that inhibiting the mTOR pathway will sensitize breast cancers expressing EGFR to gefitinib inhibition. In summary, these data provide strong evidence for the presence of a de novo EGFR TKI resistance pathway in breast cancers that occurs through the activation of the mTOR pathway and therapy for EGFR positive breast cancers could be improved with dual treatment of EGFR TKI and an mTOR antagonist. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1068. doi:1538-7445.AM2012-1068