HER2/ErbB2 receptor signaling in rat and human prolactinoma cells: strategy for targeted prolactinoma therapy

Dopamine agonist resistance or intolerance is encountered in approximately 20% of prolactinoma patients. Because human epidermal growth factor receptor 2 (HER2)/ErbB2 is overexpressed in prolactinomas and ErbB receptor ligands regulate prolactin (PRL) gene expression, we tested the role of HER2/ErbB2 in prolactinoma hormone regulation and adenoma cell proliferation to assess the rationale for targeting this receptor for prolactinoma therapy. As we showed prolactinoma HER2 overexpression, we generated constitutively active HER2-stable GH3 cell transfectants (HER2CA). PRL mRNA levels were induced approximately 250-fold and PRL secretion was enhanced 100-fold in HER2CA cells, which also exhibited increased proliferation. Lapatinib, a dual tyrosine kinase inhibitor (TKI) of both epidermal growth factor receptor (EGFR)/ErbB1 and HER2, blocked receptor signaling, and suppressed PRL expression more than gefitinib, a TKI of EGFR/ErbB1. Lapatinib also suppressed colony formation in soft agar more than gefitinib. Oral lapatinib treatment caused tumor shrinkage and serum PRL suppression both in HER2CA transfectant-inoculated Wistar-Furth rats and in estrogen-induced Fischer344 rat prolactinomas. In cultured human cells derived from resected prolactinoma tissue, lapatinib suppressed both PRL mRNA expression and secretion. These results demonstrate that prolactinoma HER2 potently induces PRL and regulates experimental prolactinoma cell proliferation. Because pituitary HER2 signaling is abrogated by TKIs, this receptor could be an effective target for prolactinoma therapy.     

The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation

Background

Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation.

Results and Discussion

Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4’-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1–10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.

Conclusion

Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.     

Simultaneous Inhibition of Estrogen Receptor and the HER2 Pathway in Breast Cancer: Effects of HER2 Abundance

The estrogen receptor (ER) pathway and the epidermal growth factor receptor (EGFR) pathway play pivotal roles in breast cancer progression. Targeted therapies able to intercept ER or signaling downstream to EGFR and its kin, HER2, are routinely used to treat distinct groups of breast cancer patients. However, patient responses are limited by resistance to endocrine therapy, which may be due to compensatory HER2/EGFR signaling. This raises the possibility that simultaneous interception of HER2 and ER may enhance therapeutic efficacy. To address the question, we treated breast cancer cells with both fulvestrant (ICI 182780), an ER antagonist with no agonist effects, and lapatinib, an orally available tyrosine kinase inhibitor specific to EGFR and HER2. Our results indicate that the combination of drugs is especially effective when applied to HER2-overexpressing, ER-positive cancer cells. Interestingly, fulvestrant activated the mitogen-activated protein kinase (MAPK) pathway of these cells, but complete inhibition of MAPK signaling was observed on cotreatment with lapatinib. Taken together, our observations reinforce the possibility that the effectiveness of combining anti-ER and anti-HER2/EGFR drugs may be especially effective on a relatively small subtype of HER2-overexpressing, ER-positive tumors of the breast.     

Mechanisms of resistance to HER family targeting antibodies

The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.     

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ET_A receptor (R) and ET_BR on cancer cells. High levels of tumor ET-1 and ET_AR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ET_AR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ET_AR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca²⁺ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.     

Tumor-derived lactate induces M2 macrophage polarization via the activation of the ERK/STAT3 signaling pathway in breast cancer

Tumor-associated macrophages (TAM) are prominent components of tumor microenvironment (TME) and capable of promoting cancer progression. However, the mechanisms for the formation of M2-like TAMs remain enigmatic. Here, we show that lactate is a pivotal oncometabolite in the TME that drives macrophage M2-polarization to promote breast cancer proliferation, migration, and angiogenesis. In addition, we identified that the activation of ERK/STAT3, major signaling molecules in the lactate signaling pathway, deepens our molecular understanding of how lactate educates TAMs. Moreover, suppression of ERK/STAT3 signaling diminished tumor growth and angiogenesis by abolishing lactate-induced M2 macrophage polarization. Finally, research data of the natural compound withanolide D provide evidence for ERK/STAT3 signaling as a potential therapeutic strategy for the prevention and treatment of breast cancer. These findings suggest that the lactate-ERK/STAT3 signaling pathway is a driver of breast cancer progression by stimulating macrophage M2-like polarization and reveal potential new therapeutic targets for breast cancer treatment.     

Lapatinib,a dual HER1/HER2 tyrosine kinase inhibitor,augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2‐driven cancer cell growth

The ultimate biological and clinical meaning of shed HER2 extracellular domain (ECD) has remained largely unclear until recently. Oversecretion of soluble HER2 ECD has been shown to inhibit growth of HER2‐overexpressing cancer cells by promoting HER2 ECD dimerization with HER transmembrane receptors thus impairing their cross‐tyrosine phosphorylation and decreasing their activation status. HER2‐targeted drugs capable to enhance the occurrence of basal HER2 ECD shedding but simultaneously preventing formation of truncated cell membrane‐bound HER2 intracellular fragment, which exhibits an undesirable constitutive kinase activity, might be extremely efficient at managing HER2‐positive cancer disease. The dual HER1/HER2 Tyrosine Kinase inhibitor lapatinib, which works intracellularly and directly targets the TK domain of HER2, drastically augments basal shedding of HER2 ECD to inhibit HER2‐driven cancer cell growth. Lapatinib treatment significantly augments the concentration of the inactive (unphosphorylated) form of HER2 protein at the tumor cell membrane and promotes an exacerbated HER2 ECD shedding to the extracellular milieu of HER2‐overexpressing cancer cells. Exacerbated sensitivity of trastuzumab‐resistant cancer cells, which contain nearly undetectable levels of soluble HER2 ECD when compared with trastuzumab‐sensitive parental cells to lapatinib‐induced cell growth inhibition, takes place when lapatinib treatment fully restores high levels of basal HER2 ECD shedding. The dramatic augmentation of HER2 ECD shedding that occurs upon treatment of with lapatinib is fully suppressed in lapatinib‐refractory HER2‐positive cells. These findings, altogether, may provide crucial insights concerning clinical studies aimed to accurately describe HER2 ECD as a potential predictor of response or resistance to the HER2‐targeted drugs trastuzumab and lapatinib. J. Cell. Physiol. 226: 52–57, 2010. © 2010 Wiley‐Liss, Inc.     

Src and CXCR4 are involved in the invasiveness of breast cancer cells with acquired resistance to lapatinib

Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.     

The docking molecule gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common gamma chain-using cytokines

Interleukin (IL)-2, a critical cytokine with indispensable functions in regulating lymphoid homeostasis, induces the activation of several biochemical pathways. Precisely how these pathways are linked and how they relate to the biological action of IL-2 is incompletely understood. We previously identified SHP-2 (Src homology 2 domain containing phosphatase 2) as an important intermediate in IL-2-dependent MAPK activation and showed its association with a 98-kDa phosphoprotein in response to IL-2. Here, we demonstrate that Gab2, a recently identified adapter molecule, is the major SHP-2 and phosphatidylinositol 3'-kinase-associated 98-kDa protein in normal, IL-2-activated lymphocytes. We further demonstrate that phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2 receptor beta chain. Gab2 can be a substrate of all the three major classes of non-receptor tyrosine kinases associated with the IL-2R, but in terms of IL-2 signaling, JAK3 but not Lck or Syk is essential for Gab2 phosphorylation. We also demonstrate that only IL-2 and IL-15, but not other gammac cytokines induce Gab2 phosphorylation; the ability to phosphorylate Gab2 correlates with Shc phosphorylation and ERK1/ERK2 activation. Finally, we also show that Gab2 levels are regulated by T cell activation, and resting T cells express little Gab2. Therefore, up-regulation and activation of Gab2 may be important in linking the IL-2 receptor to activation of MAPK and may be an important means of achieving specificity in cytokine signaling.     

Lapatinib,a Dual EGFR and HER2 Tyrosine Kinase Inhibitor,Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

Background

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated.

Methodology and Principal Findings

In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib.

Conclusions and Significance

These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine.     

Control of FLIPL expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP_L) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP_L levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP_L upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP_L expression. FLIP_L upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP_L upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP_L levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.     

Src family kinase-independent signal transduction and gene induction by leukemia inhibitory factor

Members of the interleukin-6 (IL-6) family of cytokines exert their biological effects via binding to their cognate ligand-binding receptor subunit on a target cell. The subsequent recruitment of the common signal transducer glycoprotein 130 and activation of the JAK/STAT and SHP-2/Ras/mitogen-activated protein kinase (MAPK) pathways are responsible for the majority of cellular responses elicited by IL-6 cytokines. Several types of experiments suggest that the Src family of kinases (SFK) also participates in IL-6 family cytokine-mediated signaling events. SYF cells, which lack expression of SFKs Src, Yes, and Fyn, were used to determine the role of SFKs in IL-6 family cytokine signaling and gene induction. SYF and wild type (WT) control fibroblasts displayed similar activation of signaling intermediates following stimulation with leukemia inhibitory factor (LIF). LIF-stimulated tyrosine phosphorylation of SHP-2 and subsequent activation of MAPK in SYF cells were identical to that seen in LIF-stimulated WT cells. Both LIF-stimulated tyrosine phosphorylation of STAT1 and STAT3, as well as LIF-stimulated DNA binding activity of STAT-containing nuclear complexes were indistinguishable when compared in SYF and WT cells. In addition, the phosphatidylinositol 3-kinase-sensitive Akt kinase and p38 MAPK were activated by LIF in both SYF and WT cells. Furthermore, LIF-stimulated expression of c-fos, egr-1, and suppressor of cytokine signaling-3 was retained in SYF cells. The IL-6 family cytokine oncostatin M was also capable of activating MAPK, STAT3, STAT1, Akt, and p38 in both WT and SYF cells. These results demonstrate that IL-6 family cytokines can activate a full repertoire of signaling pathways and induce gene expression independent of SFKs.     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.     

t-Darpp Promotes Enhanced EGFR Activation and New Drug Synergies in Her2-Positive Breast Cancer Cells

Trastuzumab has led to improved survival rates of HER2⁺ breast cancer patients. However, acquired resistance remains a problem in the majority of cases. t-Darpp is over-expressed in trastuzumab-resistant cell lines and its over-expression is sufficient for conferring the resistance phenotype. Although its mechanism of action is unknown, t-Darpp has been shown to increase cellular proliferation and inhibit apoptosis. We have reported that trastuzumab-resistant BT.Her^R cells that over-express endogenous t-Darpp are sensitized to EGFR inhibition in the presence (but not the absence) of trastuzumab. The purpose of the current study was to determine if t-Darpp might modulate sensitivity to EGFR inhibitors in trastuzumab-resistant cells. Using EGFR tyrosine kinase inhibitors AG1478, gefitinib and erlotinib, we found that trastuzumab-resistant SK.Her^R cells were sensitized to EGFR inhibition, compared to SK-Br-3 controls, even in the absence of trastuzumab. t-Darpp knock-down in SK.Her^R cells reversed their sensitivity to EGFR inhibition. Increased EGFR sensitivity was also noted in SK.tDp cells that stably over-express t-Darpp. High levels of synergy between trastuzumab and the EGFR inhibitors were observed in all cell lines with high t-Darpp expression. These cells also demonstrated more robust activation of EGFR signaling and showed greater EGFR stability than parental cells. The T75A phosphorylation mutant of t-Darpp did not confer sensitivity to EGFR inhibition nor activation of EGFR signaling. The over-expression of t-Darpp might facilitate enhanced EGFR signaling as part of the trastuzumab resistance phenotype. This study suggests that the presence of t-Darpp in HER2⁺ cancers might predict the enhanced response to dual HER2/EGFR targeting.     

Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase Cdelta, Lyn kinase, and matrix metalloproteinases

We have demonstrated earlier that lysophosphatidic acid (LPA)-induced interleukin-8 (IL-8) secretion is regulated by protein kinase Cdelta (PKCdelta)-dependent NF-kappaB activation in human bronchial epithelial cells (HBEpCs). Here we provide evidence for signaling pathways that regulate LPA-mediated transactivation of epidermal growth factor receptor (EGFR) and the role of cross-talk between G-protein-coupled receptors and receptor-tyrosine kinases in IL-8 secretion in HBEpCs. Treatment of HBEpCs with LPA stimulated tyrosine phosphorylation of EGFR, which was attenuated by matrix metalloproteinase (MMP) inhibitor (GM6001), heparin binding (HB)-EGF inhibitor (CRM 197), and HB-EGF neutralizing antibody. Overexpression of dominant negative PKCdelta or pretreatment with a PKCdelta inhibitor (rottlerin) or Src kinase family inhibitor (PP2) partially blocked LPA-induced MMP activation, proHB-EGF shedding, and EGFR tyrosine phosphorylation. Down-regulation of Lyn kinase, but not Src kinase, by specific small interfering RNA mitigated LPA-induced MMP activation, proHB-EGF shedding, and EGFR phosphorylation. In addition, overexpression of dominant negative PKCdelta blocked LPA-induced phosphorylation and translocation of Lyn kinase to the plasma membrane. Furthermore, down-regulation of EGFR by EGFR small interfering RNA or pretreatment of cells with EGFR inhibitors AG1478 and PD158780 almost completely blocked LPA-dependent EGFR phosphorylation and partially attenuated IL-8 secretion, respectively. These results demonstrate that LPA-induced IL-8 secretion is partly dependent on EGFR transactivation regulated by PKCdelta-dependent activation of Lyn kinase and MMPs and proHB-EGF shedding, suggesting a novel mechanism of cross-talk and interaction between G-protein-coupled receptors and receptor-tyrosine kinases in HBEpCs.