ABSTRACT
Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/ß-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the ß-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/ß-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.
Subject(s)
ErbB Receptors/metabolism , Hair Follicle/growth & development , Hair Follicle/metabolism , Wnt Signaling Pathway , Animals , Apoptosis/drug effects , Cell Count , Cell Differentiation/drug effects , Cell Proliferation/drug effects , DNA Damage , Gene Deletion , Gene Expression Regulation, Developmental/drug effects , Gene Knock-In Techniques , Hair/growth & development , Hair Follicle/cytology , Ligands , Mice , Mitosis/drug effects , Models, Biological , Morphogenesis/drug effects , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Skin/growth & development , Transcription, Genetic/drug effects , Transforming Growth Factor alpha/pharmacology , Wnt Signaling Pathway/drug effects , ras Proteins/metabolismABSTRACT
Our previous report has shown that the constitutively activated EGFR variant, EGFRvIII, up-regulates the pro-metastatic chemokine receptor CXCR4 in breast cancer cells. Here we evaluated the biological effect and cell signaling effects of silencing CXCR4 expression in EGFRvIII-expressing breast cancer cells. Short hairpin RNA (shRNA)-mediated suppression of CXCR4 expression significantly reduced the invasive potential and proliferation of EGFRvIII-expressing breast cancer cells. These cells exhibited a reduction of EGFRvIII activity and protein expression due to increased protein degradation and altered protein trafficking. In conclusion, suppression of CXCR4 inhibits EGFRvIII-mediated breast cancer cell invasion and proliferation.
Subject(s)
Breast Neoplasms/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Receptors, CXCR4/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Separation , Female , Flow Cytometry , Gene Silencing , Humans , Models, Biological , Neoplasm Invasiveness , Proteasome Endopeptidase Complex/metabolism , Protein TransportABSTRACT
Epidermal Growth Factor Receptor (EGFR) mutants are associated with resistance to chemotherapy, radiation, and targeted therapies. Here we found that the phytochemical 3,3'-Diindolylmethane (DIM) can inhibit the growth and also the invasion of breast cancer, glioma, and non-small cell lung cancer cells regardless of which EGFR mutant is expressed and the drug-resistant phenotype. DIM reduced an array of growth factor signaling pathways and altered cell cycle regulators and apoptotic proteins favoring cell cycle arrest and apoptosis. Therefore, DIM may be used in treatment regimens to inhibit cancer cell growth and invasion, and potentially overcome EGFR mutant-associated drug resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm , ErbB Receptors/metabolism , Glioma/drug therapy , Indoles/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/drug effects , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , ErbB Receptors/genetics , Female , Genes, erbB-1 , Glioma/genetics , Glioma/metabolism , Glioma/pathology , Humans , Indoles/therapeutic use , Mutant Proteins/metabolism , Mutation , Neoplasm Invasiveness , Phosphorylation , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Stress, PhysiologicalABSTRACT
The expression of the potent, constitutively activated EGFR variant, EGFRvIII, has been linked to breast cancer metastasis, but the mechanisms of EGFRvIII and CXCR4 crosstalk, which may facilitate breast cancer invasion, have never been explored. Here we report that CXCR4 expression is increased in breast cancer cells expressing EGFRvIII regardless of the ER/PgR status of the cells. Treatment of EGFRvIII-expressing breast cancer cells with the tyrosine kinase inhibitor, AG1478, reverses CXCR4 expression back to levels expressed in parental cells. In addition, expressing EGFRvIII enhances CXCL12/CXCR4-mediated invasion, which can be inhibited by CXCR4 inhibitors. Surprisingly, CXCR4 mRNA and its transcriptional regulator, HIF-1alpha, are up-regulated only in ER+/PgR+ estrogen-dependent EGFRvIII-expressing breast cancer cells, but not in ER-/PgR- or estrogen-independent cell lines, suggesting that HIF-1alpha and hormone receptor-mediated actions may have a role in the transcriptional regulation of CXCR4. We also demonstrate that p38 MAPK is one of the major down-stream signaling molecules responsible for EGFRvIII/CXCR4-mediated invasion as p38 MAPK activity was induced by CXCL12 stimulation under both normoxic and hypoxic conditions. More interestingly, inhibition of p38 MAPK activity significantly reduced CXCR4 expression and inhibited the invasive potential of EGFRvIII-expressing breast cancer cells, suggesting an essential role for p38 MAPK in EGFRvIII/CXCR4 induced invasion. Furthermore, CXCR4 is regulated post-translationally through decreased expression of AIP4 and beta-arrestin 1/2, molecules involved in CXCR4 internalization, cellular trafficking and degradation. These results provide a plausible mechanism for EGFRvIII-mediated invasion and establish a functional link between EGFRvIII and CXCR4 signaling pathways.
Subject(s)
Breast Neoplasms/metabolism , ErbB Receptors/genetics , Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness/genetics , Receptors, CXCR4/biosynthesis , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Separation , ErbB Receptors/metabolism , Female , Flow Cytometry , Gene Expression , Humans , Immunoblotting , Mice , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Processing, Post-Translational , Receptors, CXCR4/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Transcription, GeneticABSTRACT
The tumor-specific, ligand-independent, constitutively active epidermal growth factor receptor (EGFR) variant, EGFRvIII, remains understudied in breast cancer. Here, we report that expression of EGFRvIII in the ErbB-2-overexpressing, estrogen-dependent MDA-MB-361 breast cancer cell line resulted in significant estrogen-independent tumor growth in ovariectomized, athymic nude mice in comparison to MDA-MB-361/wt cells. MDA-MB-361/vIII breast cancer cells maintained estrogen-induced tumor growth, but were tamoxifen-resistant in the presence of estrogen, while MDA-MB-361/wt cells had a significant reduction in tumor growth in the presence of estrogen and tamoxifen. Tamoxifen alone did not have a significant effect on EGFRvIII-mediated estrogen-independent tumor growth. Constitutive signaling from the EGFRvIII receptor resulted in an increased activation of both the Akt and MAPK pathways. Compared to estrogen-dependent, tamoxifen-sensitive MCF-7/vIII breast cancer cells, which had unchanged levels of ERalpha, but an increase in progesterone receptor (PgR) in comparison to MCF-7/wt cells, MDA-MB-361/vIII cells had a reduction in ERalpha expression as well as a more pronounced reduction in PgR compared with MDA-MB-361/wt cells. EGFRvIII expression was also significantly associated with an absence of PgR protein in invasive human breast cancer specimens. Alterations of proapoptotic proteins and antiapoptotic proteins were observed in EGFRvIII transfectants. In conclusion, constitutive signaling through EGFRvIII and its downstream effector proteins crosstalks with the ERalpha pathway, resulting in loss of PgR expression and alterations in the apoptotic pathway, which may result in the estrogen-independent, tamoxifen-resistant phenotype conferred to EGFRvIII-expressing breast cancer cells.
