| Background and objectiveThe epidermal growth factor receptor (EGFR) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. The epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases:EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). Mutations affecting EGFR expression or activity could result in cancer.Estrogen receptors (ER) are a group of proteins found inside cells. They are receptors that are activated by the hormone estrogen (17β-estradiol). Once activated by estrogen, the ER is able to translocate into the nucleus and bind to DNA to regulate the activity of different genes (i.e. it is a DNA-binding transcription factor). However, it also has additional functions independent of DNA binding.Protein tyrosine phosphatase H1 (PTPH1), also called Tyrosine-protein phosphatase non-receptor type 3 (PTPN3) is an enzyme that in humans is encoded by the PTPN3 gene. The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation.A tyrosine-kinase inhibitor (TKI) is a pharmaceutical drug that inhibits tyrosine kinases. Tyrosine kinases are enzymes responsible for the activation of many proteins by signal transduction cascades. The proteins are activated by adding a phosphate group to the protein (phosphorylation). TKIs are typically used as anti-cancer drugs. Studies from our lab have previously shown that PTPHl dephosphorylate EGFR/Y1173. Moreover, our lab published studies showing that PTPH1 can increase breast cancer invasion. Importantly, PTPH1 protein also increases breast cancer sensitivity to Tamoxifen. These studies together suggest that PTPHl may be an important factor to regulate anti-tumor drug sensitivity in breast cancer cells.MethodsTo test our hypothesis, we designed a three-part plan of test.In the first part of the experiment, we selected PTPH1 overexpressing cancer cells with normal PTPH1 expression level cancer cell lines and PTPH1 decreased expression of cells culturing with anticancer drug, the sensitivity related experiment is for detect the impact of PTPH1 to anti-cancer drugs.In the second part of the experiment, the relevant conditional cultured cell lines, followed by Western blot to prove if PTPH1 can increase the sensitivity of anti-EGFR related anti-cancer drugs (TKI). Finally, the third part of the experiment, we designed a series of comprehensive experiments, including Western blot, immunoprecipitation, cell fractiation experiments, in which the interaction of ER and EGFR with PTPH1 regulation was showed. These experiments demonstrated PTPH1 stabilizes EGFR protein and promotes ER transfering to the nucleus, so as to enhance therapeutic target activity of breast cancer.Results1. PTPH1 positively regulates breast cancer sensitivity to tyrosine kinase inhibitors (TKIs).Small molecule TKIs are believed to inhibit cancer growth by suppressing EGFR tyrosine phosphorylation at multiple residues. It is not known, however, whether a protein tyrosine phosphatase can impact the sensitivity to this class of inhibitors by catalyzing EGFR tyrosine dephosphorylation. Since PTPH1 inhibits the EGFR/Y1173 phosphorylation, we next examined if it may regulate breast cancer sensitivity to TKIs. PTPH1 was overexpressed in MCF-7 cells by a tetracycline-inducible system (Tet-on) and in T47D cells by stable retroviral infection. To stably deplete endogenous PTPH1 proteins, cells were infected with lentivirus expressing either shLuc (control) or shRNAs targeting two separate PTPH1 sequences, followed by antibiotic selection. Cells, with or without PTPH1 overexpression or depletion, were then incubated with lapatinib (Lap), an EGFR/Her-2 dual inhibitor currently used in clinical trials. Effects on breast cancer cell growth were assessed by colony formation. Results show that PTPH1 overexpression enhances the Lap-induced growth inhibition in both cell lines, whereas its depletion resulted in an opposite effect. A similar effect was also demonstrated in 231 breast cancer cells. Moreover, PTPH1-induced sensitization was demonstrated with another TKI gefitinib (Gef), a specific EGFR inhibitor that has also been used clinically. These results together demonstrated that PTPH1 is a novel determinant of breast cancer sensitivity to targeted therapies with TKIs.2. PTPH1 dephosphorylates EGFR/Y1173 in breast cancer cells. Tyrosine phosphorylation plays an essential role in EGFR signaling to downstream mitogenic pathways and in cancer sensitivity to TKIs. Our previous studies demonstrated that the tyrosine phosphatase PTPH1 dephosphorylates EGFR at Y1173 in vitro. We therefore first determined if PTPH1 affects EGFR tyrosine phosphorylation in breast cancer cells. Results showed that stable PTPH1 expression in T47D breast cancer cells decreases levels of endogenous and EGF-induced tyrosine phosphorylation of EGFR at Y1173 (p-EGFR/Y1173). Moreover, knockdown of endogenous PTPH1 by two separate shRNAs increases P-EGFR/Y1173 levels with and without EGF treatment. PTPH1 also similarly regulates p-EGFR/Y1173 expression in MCF-7 breast cancer cells. PTPH1 specifically dephosphorylates EGFR on Y1173 but not on Y1168. These results indicate an active role of PTPH1 in control of p-EGFR/Y1173 expression in breast cancer cells. We previously showed that p38 MAPK phosphorylates PTPH1/S459, which is required for PTPH1 to increase Ras-dependent growth and to inhibit stress-induced cell death. We next examined if S459 is required for the PTPH1 catalytic activity to dephosphorylate EGFR/Y1173 as compared with the positive control PTPH1/DA (a phosphatase-deficient trapping mutant). Transient PTPH1 expression significantly decreases levels of the co-expressed EGFR phosphorylation at Y1173, whereas neither of its mutants has such effect. Together, these results demonstrate that PTPH1 efficiently catalyzes Tyr1173 dephosphorylation of EGFR, which may play a critical role in regulating the therapeutic target activity of EGFR.3.PTPH1 increases EGFR protein expression by stabilization. Tyrosine phosphorylation typically triggers EGFR degradation through an internalization process. Analysis of whole cell lysates showed that PTPH1 expression increases endogenous and transfected EGFR protein expression. We next examined if PTPH1 increased EGFR protein stability by decreasing its phosphorylation at Y1173. T47D cells stably expressed with PTPH1 and its phosphatase-inactive PTPH1/S459A mutant were cultured with cycloheximide (CHX), a protein synthesis inhibitor. Endogenous EGFR protein expression was examined by direct WB analysis. Results show that PTPH1 significantly increases the EGFR stability as compared to its S459A mutant, indicating that PTPH1 depends on its catalytic activity to stabilize EGFR protein. Analysis of MCF-7 cells further demonstrated that PTPHl-forced expression stabilizes the ectopically expressed EGFR but not EGFR/Y1173, and the EGFR/Y1173F is more stable than its WT counterpart. Transient co-transfection in 293T cells further demonstrated that PTPH1 inhibits EGFR (but not its Y1173F mutant) ubiquitination and proteasome-dependent degradation, whereas PTPH1/S459A mutant is less effective in these actions.In addition, elevated PTPH1 in breast cancer tissues correlates with increased EGFR protein expression. These results together demonstrate that PTPH1 increases EGFR protein expression through decreasing its phosphorylation at Y1173 and thereby inhibiting its proteasome-dependent degradation.4. PTPH1 increases the breast cancer sensitivity by disrupting the EGFR-ER interaction. We previously demonstrated that PTPH1 confers breast cancer anti-hormone sensitivity through inducing ER/Y537 dephosphorylation. Since PTPH1 decreases EGFR/Y1173 phosphorylation, we determined if PTPH1 requires its catalytic activity to sensitize breast cancer cells to TKIs. T47D cells stably expressed with PTPHls were assessed for the TKI-induced growth inhibition as described above. Interestingly, we found that only expressed PTPH1, but not its phosphatase-deficient mutants, significantly decreases levels of p-EGFR/Y1173 expression and significantly increases the growth-inhibition by both inhibitors. These results indicate that PTPH1 depends on its catalytic activity to sensitize breast cancer cells to TKIs. Because EGFR-ER interaction is associated with TAM resistance in breast cancer and EGFR/ER signal cross-talk is bidirectional, we next tested if PTPH1 enhances TKI-induced growth-inhibition by regulating the EGFR-ER binding. WB analyses of either anti-EGFR or ant-ER immunoprecipitates revealed their complex-formation as previously reported. This complex however is disrupted by PTPH1 overexpression as demonstrated in EGFR precipitates, but not by its mutant S459A, indicating an inhibition of the complex by tyrosine dephosphorylation. Consistent with our previous findings, cell fractionation analysis showed that PTPH1 depends on its phosphatase activity to increase ER nuclear accumulation. Interestingly, PTPH1 also stimulates EGFR protein expression, especially in cytoplasmic compartment. It is of interest to note that PTPH1 proteins are also detectable in their precipitates and an inhibition of the EGFR-ER interaction in PTPH1 expressed cells couples with its relocation from the ER precipitates to the EGFR complexes. Since the tyrosine kinase EGFR is considered to be a natural substrate of tyrosine phosphatases such as PTPH1, one mechanism for the disruption of the EGFR-ER interaction may be due to the fact that the ectopically expressed PTPH1 competes with and consequently replaces endogenous ER for interaction with EGFR. This conclusion is further supported by increased EGFR but decreased ER proteins in the PTPH1 precipitates in PTPH1 overexpressed cells as compared to those transfected with vector, albeit the effect on ER being less substantial. These effects require the PTPH1 catalytic activity and are associated with the increased sensitivity to TKIs. These results together indicate that PTPH1 increases the growth-inhibition of TKIs by disrupting the EGFR-ER interaction through its EGFR binding activity via a competitive enzyme-substrate interaction.5. Expression of a nuclear-localized defective ER alone is sufficient to enhance its interaction with EGFR and to confer the resistance to TKIs. One explanation for the PTPH1 capacity to disrupt the EGFR/ER complex may be due to its stimulation of ER nuclear accumulation as a result of the ER/Y537 dephosphorylation. This would lead to decreased levels of extra-nuclear ER proteins available for interacting with cytoplasmic EGFR. To demonstrate if an alteration of cellular ER localization alone is sufficient to impact its interaction with EGFR, we used the Tet-on system to express ER and its mutant ER/T311A in ER negative 231 cells. Thr311 in the hormone-binding domain of ER is required for ER nuclear localization and its mutation to Ala reduces ER nuclear levels. Results showed that although the ER/T311A is expressed to a lesser extent than wild-type (WT) ER after Tet addition in whole cell lysates (WCL), its relative level in the cytoplasm over the nucleus is higher than that of the WT ER. Analysis of anti-EGFR precipitates show a greater amount of the EGFR-ER complex-formation in ER/T311A than WT ER expressed cells, indicating that the cytoplasmic ER has a higher binding affinity to EGFR protein. Consistent with this conclusion, the cytoplasmic PTPH1 also binds more ER/T311A than ER. Because ER and ER/T311A are expressed at different levels after Tet addition {likely as a result of their different stability and/or different localizations}, they were transiently co-transfected with Myc-EGFR in 293T cells to further compare their EGFR binding activities. WB analysis of the Myc precipitates showed that Myc-EGFR binds increased levels of the cytoplasmic ER/T311A but decreased amounts of the nuclear GFP-ER/Y537F as compared to their respective WT proteins under the conditions where ER and its mutants are expressed at similar levels. These results further demonstrate that EGFR has a higher binding affinity to the cytoplasmic ER but decreased activity in interaction with, the nuclear ER. Importantly, ER/T311A expressed cells are more resistant to both TKIs than those expressed with WT ER. These results further demonstrate that the ER-bound EGFR is less effective than its free form as a therapeutic target for TKI-induced growth inhibition.6. PTPH1 increases the membranous EGFR and nuclear ER levels, and confers breast cancer sensitivity to a combined therapy of TKIs with an anti-estrogen. PTPH1 belongs to the non-receptor PTP family and is mostly localized to the cytosol. EGFR is a transmembrane receptor and an EGFR translocation to the nucleus is associated with resistance to EGFR targeted therapies whereas its membranous accumulation appears to be necessary for anti-EGFR therapy. In contrast, the nuclear receptor ER exerts its biological functions both through its nuclear and extra-nuclear activities. Our results showed that PTPH1 positively regulates breast cancer sensitivities to TKIs through decreasing EGFR/Y1173 phosphorylation and disrupting the ER-EGFR interaction. These results, together with PTPH1 stimulation of ER nuclear accumulation and of breast cancer sensitivity to anti-estrogens, indicate that a physical interaction between EGFR and ER in low PTPH1 expressed cells may restrain or limit their therapeutic target activities through an alteration of their intrinsic cellular localization. Higher concentrations of cellular PTPH1 proteins may then restore the natural EGFR and ER cellular distributions by attenuating their complex-formation and consequently confer the sensitivity to combined therapies of TKIs with an anti-estrogen. To test this possibility, T47D cells with and without stably expressed PTPH1 (or its mutant) were analyzed for their sensitivity to combined therapies of TKIs with the ER inhibitor tamoxifen (TAM). To further dissect the distribution of EGFR and ER in different cellular compartments, a recently published protocol was used to prepare proteins from the membrane, cytosol, and nucleus. Results showed an increased growth-inhibition by the combined treatment of Lap or Gef with TAM in Vector-transfected cells over either inhibitor alone. A forced PTPH1 (but not PTPH1/S459A) expression significantly increases the growth-inhibition by the combination of inhibitors compared to either alone. Cell fractionation analysis showed that the PTPH1 expression stimulates both the membranous EGFR and the nuclear ER accumulation, while its catalytic deficient mutant lacks such activities. Similar results were also obtained in MCF-7 cells. The sensitization effect of PTPH1 to the combined therapy was further demonstrated by cell viability assays. Experiments with stably co-transfected MCF-7 cells further showed that PTPHl increased the membranous EGFR and the nuclear ER as demonstrated by cell fractionation and immune-staining analyses. Moreover, elevated PTPHl protein expression in breast cancer tissues appears to couple with a more dramatic increase in the membranous EGFR expression. These results together suggest that PTPH1 may confer breast cancer sensitivity to combined therapies of TKIs with TAM by restoring EGFR and ER physiological localizations through disrupting their complex-formation via decreasing their tyrosine phosphorylation.Conclusions1. PTPH1 increases breast cancer cells sensitivity to TKI.2. PTPH1 dephosphorylates EGFR/Y1173 in vitro and in breast cancer cells.3. PTPH1 increases EGFR cytosolic stability via dephosphorylates EGFR/Y1173.4. PTPH1 depends on its catalytic activity to decrease EGFR-ER complex so to enhance TKI with ER inhibitor treatment of breast cancer.SignificanceEGFR plays a determinant role in breast cancer drug sensitivity. Our results presented here have advanced this field by demonstrating that EGFR dephosphorylation at a critical residue such as Y1173 is equally important for breast cancer response to Lapatinib therapy through increasing EGFR stability. First, we showed that PTPH1 dephosphorylates EGFR/Y1173 in vitro and in breast cancer cells thus acting as a novel EGFR phosphatase. Further, PTPH1 was demonstrated to enhance EGFR protein stability and to increase EGFR cytosol localization depending on Y1173. These results indicate a role of PTPHl-induced and Y1173-dependent EGFR protein turnover that may be involved in Lapatinib drug resistance. Most importantly, PTPH1 increases breast cancer TKI sensitivity in cell culture, and requires its catalytic activity and/or Y1173 to increase EGFR cytosol localization and EGFR protein level, and/or to enhance the TKI-induced growth-inhibition. These results together indicate that it is PTPH1-induced ER/Y1173 dephosphorylation that integrates the EGFR stability with the enhanced LAP sensitivity. Because EGFR gene expression in clinical breast cancer correlates with TKI therapy, our results provide the critical experimental evidence to indicate an application potential of increasing EGFR stability by PTPH1 in increasing breast cancer TKI sensitivity. This possibility is further supported by the fact that PTPHl is overexpressed in about 50% of clinical breast cancer. Targeting EGFR/Y1173 de-phosphorylation by PTPH1 so as to upregulate cytosol EGFR in breast cancer may therefore be a novel approach to improve the clinical response of TKI therapy. |
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