Autocrine Epidermal Growth Factor Receptor Research Articles

Epidermal growth factor receptor signaling suppresses αvβ6 integrin and promotes periodontal inflammation and bone loss.

In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvβ6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-β1 (TGF-β1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress β6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-β1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-β1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the β6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD.

Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development.

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)/β-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 β-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/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70–90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparinbinding EGF-like growth factor (HB-EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB-EGF had the highest mRNA expression and HB-EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB-EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB-EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB-EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB-EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB-EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulatorNfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary tomaintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer exvivo and invivo, representing a promising synthetic lethal strategy for treating the disease.

Abstract PR01: Oncogenic Kras-induced mitochondrial oxidative stress in pancreatic acini modulates the formation of precancerous lesions through autocrine EGFR signaling

Abstract The development of pancreatic cancer requires both obtain of oncogenic Kras mutations and up-regulation of growth factor signaling. However, how oncogenic Kras regulates growth factor signaling in pancreatic cancer development is still unclear. Using a transgenic mouse model harboring an oncogenic Kras mutation and a 3D organoid explant culture of primary pancreatic acini, we here show that expression of mutant Kras in pancreatic acinar cells alters their mitochondrial metabolism, resulting in increased generation of mitochondrial reactive oxygen species (mROS). We further demonstrate that mROS induced by oncogenic Kras is required for transdifferentiating acinar cells to a duct-like phenotype and eventually progression to pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesions of pancreatic ductal adenocarcinoma (PDAC). This transdiffentiation process is mediated by increased levels of epidermal growth factor receptor (EGFR) as well as its ligands, EGF and TGFα, through activation of the ROS-receptive transcription factors, NF-κB1 and NF-κB2. In Kas transgenic mice, the interception of KrasG12D-mediated generation of mROS significantly reduced the formation of pre-neoplastic lesions. Hence, our data provide mechanistic insight of how oncogenic Kras mutant crosstalks with growth factor signaling to cause the formation of pancreatic cancer. Citation Format: Geou-Yarh Liou, Heike Doeppler, Kathleen E. DelGirono, Howard C. Crawford, Michael P. Murphy, Peter Storz. Oncogenic Kras-induced mitochondrial oxidative stress in pancreatic acini modulates the formation of precancerous lesions through autocrine EGFR signaling. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR01.

Integrin α6β4 Promotes Autocrine Epidermal Growth Factor Receptor (EGFR) Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF)

Integrin α6β4 is up-regulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 up-regulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in The Cancer Genome Atlas Pancreatic Cancer Database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on hepatocyte growth factor (HGF)-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways, including MAPK, PI3K, and PKC. Moreover, matrix metalloproteinase activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional up-regulation of key EGFR family members and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.

Interleukin-1 beta transactivates epidermal growth factor receptor via the CXCL1-CXCR2 axis in oral cancer.

Hyperactivation of the epidermal growth factor receptor (EGFR) pathways and chronic inflammation are common characteristics of oral squamous cell carcinoma (OSCC). Previously, we reported that OSCC cells secrete interleukin-1 beta (IL-1β), which promotes the proliferation of the oral premalignant cell line, DOK, and stimulates DOK and OSCC cells to produce the chemokine CXCL1. CXCL1 functions through CXCR2, a G protein-coupled receptor that transactivates EGFR in ovarian and lung cancers. We hypothesized that IL-1β transactivates EGFR through the CXCL1-CXCR2 axis in OSCC. In this study, we demonstrated that tyrosine phosphorylation of EGFR is crucial for the IL-1β-mediated proliferation and subsequent bromodeoxyuridine (BrdU) incorporation of DOK cells because the EGFR inhibitors AG1478 and erlotinib inhibit these abilities in a dose-dependent manner. Addition of IL-1β instantly enhanced CXCL1 expression and secretion (within 15 min) in the DOK and OSCC cell lines. Furthermore, tyrosine phosphorylation of EGFR was significantly enhanced in DOK (1 h) and OSCC (20 min) cell lines after IL-1β treatment, and both cell lines were inhibited on the addition of an IL-1 receptor antagonist (IL-1Ra). CXCL1 treatment resulted in EGFR phosphorylation, whereas the knockdown of CXCL1 expression by lentivirus-mediated shRNA or the addition of the CXCR2 antagonist SB225002 dramatically reduced IL-1β-mediated EGFR phosphorylation and proliferation of DOK cells. Neutralizing antibodies against IL-1β or CXCL1 markedly inhibited the constitutive or IL-1β-induced tyrosine phosphorylation of EGFR in OSCC cells. IL-1β transactivates EGFR through the CXCL1-CXCR2 axis, revealing a novel molecular network in OSCC that is associated with autocrine IL-1β and EGFR signaling.

Abstract 1943: Integrin α6β4 promotes autocrine EGFR signaling to stimulate migration and invasion toward HGF

Abstract Integrin α6β4 is upregulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion, in part, through its coordinated modulation of the transcriptome. Here, we find that integrin α6β4 signaling dramatically upregulates the expression of several genes involved in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (Areg), epiregulin (Ereg), and ectodomain cleavage protease MMP1. Notably, these genes correlate well with integrin α6β4 expression in publicly available pancreatic cancer databases. We previously reported that integrin α6β4 promotes HGF-stimulated migration and invasion; therefore, we tested the role of autocrine EGFR signaling in these processes. We discovered that blocking EGFR signaling with EGFR-specific blocking antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Furthermore, we found that both Areg and Ereg are required for these autocrine effects, as knocking down either ligand prevented migration and invasion. Notably, AsPC1 cells, isolated from abdominal metastases, are not dependent on EGFR signaling for migration or invasion; thus suggesting a mechanism exists for circumventing EGFR dependency. We also demonstrated that HGF stimulates the secretion of Areg, which is dependent on integrin signaling pathways including MAPK, PI3K and PKC and that Areg was transported to the leading edge of cells treated with HGF. Moreover, MMP activity and integrin α6β4 signaling are required for Areg secretion. Finally, we found that EGFR is phosphorylated in response to HGF stimulation which was dependent on EGFR protein kinase activity; however, Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, we show that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through the upregulation of key genes and facilitating HGF-stimulated EGFR ligand secretion. Citation Format: Brittany L. Carpenter, Min Chen, Teresa Knifley, Kelley A. Davis, Susan M. Harrison, Rachel L. Stewart, Kathleen L. O'Connor. Integrin α6β4 promotes autocrine EGFR signaling to stimulate migration and invasion toward HGF. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1943. doi:10.1158/1538-7445.AM2015-1943

Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation

Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.

Autocrine-Derived Epidermal Growth Factor Receptor Ligands Contribute to Recruitment of Tumor-Associated Macrophage and Growth of Basal Breast Cancer Cells In Vivo

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.

Abstract 1647: Integrin α6β4 stimulates transcription and secretion of EGFR ligands.

Abstract Pancreatic carcinoma has the highest death to incidence ratio of all cancer due to frequent late diagnosis, drug-resistance and high metastatic potential. The integrin α6β4 is overexpressed in pancreatic carcinoma and plays an important role in tumor invasion and metastasis, in part by altering the transcriptome toward an invasive phenotype. In this study, we find that these transcriptional changes in pancreatic carcinomas include upregulation of cancer promoting genes such as ligands of the epidermal growth factor receptor (EGFR), epiregulin (Ereg) and amphiregulin (Areg). We have found previously that integrin α6β4 alters transcription by selectively targeting DNA demethylation or transcription factors, such as NFAT1 and NFAT5, to gene promoters. Given that Areg and Ereg are upregulated by over 1000-fold, we hypothesized that integrin α6β4 may control their expression by targeting their promoters for DNA demethylation. To test this concept, pancreatic cancer cells with low expression of integrin α6β4 were treated with DNA methyltransferase inhibitor 5-aza-2’deoxycytidine (DAC), and mRNA expression measured by Q-PCR. This treatment resulted in induction of Areg and Ereg expression indicating that these genes may be controlled by DNA methylation. Furthermore, suppression of integrin α6β4 by RNAi, hindered induction by DAC, thus suggesting that integrin α6β4 plays a role in ligand expression. To support these results, pancreatic cancer cells were treated with the methyl donor S-adenosylmethionine, allowing for global hypermethylation and silencing of genes controlled by methylation. Our results show that treatment inhibits Ereg and Areg expression in cells with high expression of integrin α6β4, providing evidence for epigenetic regulation by this integrin. Non-coding RNAs processed by Dicer often play a role in DNA methylation of specific genes. Accordingly, we used siRNA to target the microRNA processing enzyme Dicer. Dicer knockdown resulted in decreased expression of Ereg and Areg, measured by Q-PCR, indicating that small non-coding RNAs are involved in their regulation. Autocrine EGFR signaling is also regulated by altered secretion of Ereg and Areg. Using an ELISA for Areg, we measured secretion associated with variable expression of integrin α6β4. We find that integrin α6β4 stimulated secretion of Areg into the media, and this secretion is enhanced by HGF signaling. Consequently, we also provide evidence that integrin α6β4 promotes HGF-stimulated chemotaxis, invasion, and invasive growth which is facilitated by autocrine EGFR signaling. This study demonstrates that overexpression of integrin α6β4 in pancreatic cancer promotes a more aggressive phenotype by activating transcriptional expression and autocrine secretion of EGFR ligands, Ereg and Areg, that promote an invasive phenotype. Citation Format: Brittany L. Carpenter, Min Chen, Teresa Knifley, Kathleen L. O'Connor. Integrin α6β4 stimulates transcription and secretion of EGFR ligands. [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 1647. doi:10.1158/1538-7445.AM2013-1647

Abstract 38: The role of autocrine epidermal growth factor receptor (EGFR) signaling in breast cancer metastasis

Abstract The majority of breast cancer deaths are due to the development of metastatic disease. The metastatic cascade consists of a series of steps and includes primary tumor growth and angiogenesis, invasion, intravasation, cell survival in circulation, extravasation and sustained growth at secondary organ sites to form micrometastases. Previous studies have shown that overexpression of the epidermal growth factor receptor (EGFR; ErbB1; Her1) increases tumor cell motility, intravasation and motility. In addition, it has been demonstrated that the metastatic potential of cancer cells depends not only on their intrinsic characteristics but also on their cooperative interaction with the tumor microenvironment. For instance, imposed gradients of EGF or colony-stimulating factor-1 (CSF-1) can stimulate invasion via an EGF/CSF-1 paracrine loop between tumor cells and macrophages. Because breast malignancies exhibiting increased expression of the epidermal growth factor (EGF) family of receptors and their cognate ligands are associated with more biologically aggressive disease and poorer patient prognosis, we decided to study how expression of certain members of the EGF family of ligands in the context of ErbB1 overexpression can affect the processes of invasion, intravasation and metastasis using in vitro and in vivo systems. Based on initial Oncomine cancer gene expression analysis, heparin-binding epidermal growth factor-like growth factor (HB-EGF) overexpression was correlated with more rapid recurrence of disease in breast cancer and thus, we decided to focus our study this ligand. Briefly, MTLn3 rat mammary adenocarcinoma cells and human MDA-MB 231 breast adenocarcimona cells were transduced to overexpress HB-EGF. HB-EGF expression did not significantly affect in vitro or in vivo growth rate. In vitro microchemotaxis and invasion assays to study chemotaxis and invasion, respectively, in the presence and absence of EGF, demonstrated that random motility and spontaneous invasion were both enhanced in the presence of HB-EGF expression. Interestingly, EGF-induced chemotaxis and EGF-induced in vivo invasion were both suppressed. In vivo studies show that tumor cells expressing HB-EGF not only display increased metastasis to the lungs and extravasation from circulation, but also significantly enhanced macrophage recruitment and exerted an angiogenic effect. These findings suggest that a better understanding of the role of HB-EGF in breast cancer will aid in directing and modifying therapeutic approaches aimed at inactivating the ErbB ligand/EGFR system as well as developing approaches based on the EGF family of ligands as a target for the clinical treatment of breast cancer, ultimately leading to improved patient survival. 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 38. doi:1538-7445.AM2012-38

EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling

Epidermal growth factor (EGF) family peptides are ligands for the EGF receptor (EGFR). Here, we elucidate functional differences among EGFR ligands and mechanisms underlying these distinctions. In 32D/EGFR myeloid and MCF10A breast cells, soluble amphiregulin (AR), transforming growth factor alpha (TGFα), neuregulin 2 beta, and epigen stimulate greater EGFR coupling to cell proliferation and DNA synthesis than do EGF, betacellulin, heparin-binding EGF-like growth factor, and epiregulin. EGF competitively antagonizes AR, indicating that its functional differences reflect dissimilar intrinsic activity at EGFR. EGF stimulates much greater phosphorylation of EGFR Tyr1045 than does AR. Moreover, the EGFR Y1045F mutation and z-cbl dominant-negative mutant of the c-cbl ubiquitin ligase potentiate the effect of EGF but not of AR. Both EGF and AR stimulate phosphorylation of EGFR Tyr992. However, the EGFR Y992F mutation and phospholipase C gamma inhibitor U73122 reduce the effect of AR much more than that of EGF. Expression of TGFα in 32D/EGFR cells causes greater EGFR coupling to cell proliferation than does expression of EGF. Moreover, expression of EGF in 32D/EGFR cells causes these cells to be largely refractory to stimulation with soluble EGF. Thus, EGFR ligands are functionally distinct in models of paracrine and autocrine signaling and EGFR coupling to biological responses may be specified by competition among functionally distinct EGFR ligands.

Decreased Autocrine EGFR Signaling in Metastatic Breast Cancer Cells Inhibits Tumor Growth in Bone and Mammary Fat Pad

Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland.

Autocrine epidermal growth factor receptor ligand production and cetuximab response in head and neck squamous cell carcinoma cell lines

Predictive strategies for the treatment efficacy of cetuximab are currently not available for head and neck cancer. We investigated the correlation between the expression of epidermal growth factor receptor (EGFR) ligands and EGFR expression, and the growth inhibitory activity of cetuximab in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. The growth inhibiting effect of cetuximab was measured for eight HNSCC cell lines and correlated with the autocrine production of five EGFR ligands as measured by ELISA, and the mRNA expression of two ligands, as measured by quantitative RT-PCR. EGFR expression was assessed by western blot analysis. There was a good correlation between the expression of four of the EGFR ligands (TGF-α, amphiregulin, epiregulin and epigen) and the growth inhibiting effect of cetuximab. TGF-α had the highest predictive potential but had to be combined with epigen for full prediction. EGFR expression also correlated with cetuximab sensitivity but less clearly. The results indicate that the expression of several EGFR ligands has to be used to predict sensitivity to cetuximab in HNSCC. This has to be further evaluated in clinical samples.

Abstract 2349: Integrin α6β4 contributes to HGF-stimulated migration and invasion by promoting autocrine EGFR signaling in pancreatic cancer

Abstract The lethality of pancreatic cancer is due to elevated incidences of invasion and metastasis. Previous studies from our lab and others show that pro-invasive integrin α6β4 is upregulated in early stage pancreatic cancer, maintained during tumor progression and, in turn, promotes chemotactic migration and invasion toward HGF. To gain insight into the mechanisms governing how integrin α6β4 promotes these processes, we performed Affymetrix Gene Chip analysis on Panc-1 clones with differing levels of integrin α6β4 expression. After statistical processing, we found 582 genes altered by integrin α6β4 signaling. From these results, we found that high integrin α6β4-expressing Panc1 clones substantially enhanced expression of genes affecting epidermal growth factor receptor (EGFR) signaling. The genes upregulated by integrin α6β4 included EGFR, EGF-like ligands amphiregulin (AREG) and epiregulin (EREG), ectodomain cleavage proteases ADAMTS1, MMP1, and hyaluronan synthase 3. The upregulation of these genes was confirmed by real-time PCR, immunoblot analysis and/or ELISA. Stable overexpression of integrin β4 in low integrin β4 expressing cells increased the levels of AREG and EREG by real-time PCR. We further tested the role of autocrine EGFR signaling by pretreating cells with EGFR blocking antibodies (LA1) or EGFR-specific protein tyrosine kinase inhibitor (PD153035) and tested HGF-stimulated chemotaxis, chemo-invasion and invasive growth in three-dimensional culture. Interestingly, these inhibitors blocked integrin α6β4-dependent HGF-stimulated chemotaxis, invasion and invasive growth. In summary, our data suggest integrin α6β4 confers a motile and invasive phenotype to pancreatic carcinoma cells, in part, by coordinately regulating expression of key elements of autocrine EGFR signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2011-2349

Direct Activation of TACE-Mediated Ectodomain Shedding by p38 MAP Kinase Regulates EGF Receptor-Dependent Cell Proliferation

Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.

Reconstitution of amphiregulin-epidermal growth factor receptor signaling in lung squamous cell carcinomas activates PTHrP gene expression and contributes to cancer-mediated diseases of the bone.

Parathyroid hormone-related protein (PTHrP) is the causative factor of the paraneoplastic syndrome humoral hypercalcemia of malignancy (HHM) and it also contributes to osteolytic metastases, both of which are common complications of squamous carcinomas of the lung. Inhibition of autocrine epidermal growth factor receptor (EGFR) signaling has been shown to reduce plasma calcium and PTHrP concentrations in two lung squamous cell carcinoma xenograft models of HHM. The purpose of this study was to investigate the mechanism by which EGFR is activated and stimulates PTHrP gene expression in lung squamous carcinoma cell lines. Amphiregulin (AREG) was the only EGFR ligand that could be consistently detected in conditioned media from the SCC lines, and reduction of its expression either by siRNA or by precipitating antibody reduced PTHrP mRNA expression as effectively as EGFR-targeted inhibition. Using siRNA knockdown or inhibitors to upstream regulators of AREG shedding including TACE, Src/Lck, and G(i/o), also reduced PTHrP mRNA expression. We determined that blockade of autocrine AREG-EGFR signaling does not affect PTHrP mRNA stability. Of the three PTHrP promoters (P1, P2, and P3), P1 mRNA could be reduced by nearly 100% with an EGFR inhibitor, and both epidermal growth factor and AREG stimulated P1 mRNA by approximately 5-fold. Finally, ectopic expression of EGFR in a receptor-low but AREG-expressing cell line increased PTHrP mRNA levels in vitro, and induced the capability to cause HHM and rapid osteolytic growth in vivo. Taken together, we provide evidence that AREG stimulation of EGFR results in high levels of PTHrP gene expression, contributing to cancer-associated bone pathology.

Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

Modification of the Primary Tumor Microenvironment by Transforming Growth Factor α-Epidermal Growth Factor Receptor Signaling Promotes Metastasis in an Orthotopic Colon Cancer Model

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.