Multi-scale agent-based brain cancer modeling and prediction of TKI treatment response: Incorporating EGFR signaling pathway and angiogenesis

ABSTRACT: BACKGROUND: The epidermal growth factor receptor (EGFR) signaling pathway and angiogenesis in brain cancer act as an engine for tumor initiation, expansion and response to therapy. Since the existing literature does not have any models that investigate the impact of both angiogenesis and molecular signaling pathways on treatment, we propose a novel multi-scale, agent-based computational model that includes both angiogenesis and EGFR modules to study the response of brain cancer under tyrosine kinase inhibitors (TKIs) treatment. RESULTS: The novel angiogenesis module integrated into the agent-based tumor model is based on a set of reaction--diffusion equations that describe the spatio-temporal evolution of the distributions of micro-environmental factors such as glucose, oxygen, TGFalpha, VEGF and fibronectin. These molecular species regulate tumor growth during angiogenesis. Each tumor cell is equipped with an EGFR signaling pathway linked to a cell-cycle pathway to determine its phenotype. EGFR TKIs are delivered through the blood vessels of tumor microvasculature and the response to treatment is studied. CONCLUSIONS: Our simulations demonstrated that entire tumor growth profile is a collective behaviour of cells regulated by the EGFR signaling pathway and the cell cycle. We also found that angiogenesis has a dual effect under TKI treatment: on one hand, through neo-vasculature TKIs are delivered to decrease tumor invasion; on the other hand, the neo-vasculature can transport glucose and oxygen to tumor cells to maintain their metabolism, which results in an increase of cell survival rate in the late simulation stages.     

The Therapeutic Potential of PI3K/Akt/mTOR Inhibitors in Breast Cancer: Rational and Progress

Breast cancer (BC) is the most commonly diagnosed cancer in women. The PI3K/AKT/mTOR pathway is among the most frequently dysregulated pathways in patients with BC. The activation of this pathway is associated with increased cell growth and clinical outcome, and its overexpression is associated with a poor prognosis. It has been proposed that it may be of importance as a potential therapeutic target in the treatment of BC. The aim of current review is to provide an overview of the potential utility of PI3K/Akt/mTOR inhibitors in patients with BC, with particular emphasis on recent preclinical and clinical studies. J. Cell. Biochem. 119: 213–222, 2018. © 2017 Wiley Periodicals, Inc.     

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.Key words: EGFR, antibody synergy, functional screening, epitope binning, antibody combinations     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.Key words: EGFR, cetuximab, autophagy, apoptosis, cancer therapy     

The Epidermal Growth Factor Receptor (EGFR / HER-1) Gatekeeper Mutation T790M Is Present in European Patients with Early Breast Cancer

The epidermal growth factor receptor (EGFR) is one of the major oncogenes identified in a variety of human malignancies including breast cancer (BC). EGFR-mutations have been studied in lung cancer for some years and are established as important markers in guiding therapy with tyrosine kinase inhibitors (TKIs). In contrast, EGFR-mutations have been reported to be rare if not absent in human BC, although recent evidence has suggested a significant worldwide variation in somatic EGFR-mutations. Therefore, we investigated the presence of EGFR-mutations in 131 norwegian patients diagnosed with early breast cancer using real-time PCR methods. In the present study we identified three patients with an EGFR-T790M-mutation. The PCR-findings were confirmed by direct Sanger sequencing. Two patients had triple-negative BC (TNBC) while the third was classified as luminal-A subtype. The difference in incidence of T790M mutations comparing the TNBC subgroup with the other BC subgroups was statistical significant (P = 0.023). No other EGFR mutations were identified in the entire cohort. Interestingly, none of the patients had received any previous cancer treatment. To our best knowledge, the EGFR-T790M-TKI-resistance mutation has not been previously detected in breast cancer patients. Our findings contrast with the observations made in lung cancer patients where the EGFR-T790M-mutation is classified as a typical „second mutation”causing resistance to TKI-therapy during ongoing anticancer therapy. In conclusion, we have demonstrated for the first time that the EGFR-T790M-mutation occurs in primary human breast cancer patients. In the present study the EGFR-T790M mutation was not accompanied by any simultaneous EGFR-activating mutation.     

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

Targeting the EGFR/PCNA Signaling Suppresses Tumor Growth of Triple-Negative Breast Cancer Cells with Cell-Penetrating PCNA Peptides

Tyrosine 211 (Y211) phosphorylation of proliferation cell nuclear antigen (PCNA) coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant cells, both nuclear EGFR (nEGFR) expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC). Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP) inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP), which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC.     

Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.     

Targeted inhibition of the EGFR pathways enhances Zn‐BC‐AM PDT‐induced apoptosis in well‐differentiated nasopharyngeal carcinoma cells

Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn‐BC‐AM PDT with an EGFR inhibitor AG1478 were investigated. Well‐differentiated NPC HK‐1 cells were subjected to PDT with 1 µM of Zn‐BC‐AM and were irradiated at a light dose of 1 J/cm² in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK‐1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub‐lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn‐BC‐AM PDT in HK‐1 cells. Pre‐incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn‐BC‐AM PDT‐induced formation of apoptotic cells. The efficacy of Zn‐BC‐AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn‐BC‐AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC. J. Cell. Biochem. 108: 1356–1363, 2009. © 2009 Wiley‐Liss, Inc.     

Plakophilin-2 Promotes Tumor Development by Enhancing Ligand-Dependent and -Independent Epidermal Growth Factor Receptor Dimerization and Activation

Epidermal growth factor (EGF) receptor (EGFR) has been implicated in tumor development and invasion. Dimerization and autophosphorylation of EGFR are the critical events for EGFR activation. However, the regulation of EGF-dependent and EGF-independent dimerization and phosphorylation of EGFR has not been fully understood. Here, we report that cytoplasmic protein plakophilin-2 (PKP2) is a novel positive regulator of EGFR signaling. PKP2 specifically interacts with EGFR via its N-terminal head domain. Increased PKP2 expression enhances EGF-dependent and EGF-independent EGFR dimerization and phosphorylation. Moreover, PKP2 knockdown reduces EGFR phosphorylation and attenuates EGFR-mediated signal activation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development. Our results indicate that PKP2 is a novel activator of the EGFR signaling pathway and a potential new drug target for inhibiting tumor growth.     

EGFR signaling pathway occupies an important position in cancer‐related downstream signaling pathways of Pyk2

Proline‐rich tyrosine kinase 2 (Pyk2) is a member of focal adhesion kinase (FAK) non‐receptor tyrosine kinase family and has been found to promote cancer cell survival, proliferation, migration, invasion, and metastasis. Pyk2 takes part in different carcinogenic signaling pathways to promote cancer progression, including epidermal growth factor receptor (EGFR) signaling pathway. EGFR signaling pathway is a traditional carcinogenic signaling pathway, which plays a critical role in tumorigenesis and tumor progression. FAK inhibitors have been reported to fail to get the ideal anti‐cancer outcomes because of activation of EGFR signaling pathway. Better understanding of Pyk2 downstream targets and interconnectivity between Pyk2 and carcinogenic EGFR signaling pathway will help finding more effective targets for clinical anti‐cancer combination therapies. Thus, the interconnectivity between Pyk2 and EGFR signaling pathway, which regulates tumor development and metastasis, needs to be elucidated. In this review, we summarized the downstream targets of Pyk2 in cancers, focused on the connection between Pyk2 and EGFR signaling pathway in different cancer types, and provided a new overview of the roles of Pyk2 in EGFR signaling pathway and cancer development.     

Conformational dynamics of the EGFR kinase domain reveals structural features involved in activation

The epidermal growth factor receptor (EGFR) has been the focus of intensive studies because of its importance in cancer research. Thus, a broader understanding of the molecular mechanism of activation of the EGFR kinase will have profound significance for the development of novel therapeutics. Numerous crystal structures of EGFR kinase, including the structure of the activating‐kinase dimer, have provided snapshots of the specific pathway. Herein, we performed unrestrained‐, as well as targeted‐molecular dynamics simulations based on these data, to gain further insight into the conformational changes responsible for activation. Comparison of the monomer‐ versus activating‐EGFR‐dimer simulations indicates that the dimerization is stabilizing structural elements associated with the activated state and predicts new salt‐bridge interactions involving activation‐loop residues that may also be associated with that state. Targeted molecular dynamics simulations of the inactive‐to‐active EGFR transition, as well as the reverse pathway, confirm the formation of conserved structural features of functional importance for the activity or stabilization of either conformation. Interestingly, simulations of the L834R mutant, which is associated with cancer, suggest that the structural basis of the activation induced by that mutation might be the ability of the mutated R834 residue to consecutively form salt bridges with neighboring acidic residues and cause destabilization of a hydrophobic cluster in the inactive state. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Rab5c promotes AMAP1-PRKD2 complex formation to enhance β1 integrin recycling in EGF-induced cancer invasion

Epidermal growth factor receptor (EGFR) signaling is one of the crucial factors in breast cancer malignancy. Breast cancer cells often overexpress Arf6 and its effector, AMAP1/ASAP1/DDEF1; in these cells, EGFR signaling may activate the Arf6 pathway to induce invasion and metastasis. Active recycling of some integrins is crucial for invasion and metastasis. Here, we show that the Arf6-AMAP1 pathway links to the machinery that recycles β1 integrins, such as α3β1, to promote cell invasion upon EGFR stimulation. We found that AMAP1 had the ability to bind directly to PRKD2 and hence to make a complex with the cytoplasmic tail of the β1 subunit. Moreover, GTP-Rab5c also bound to AMAP1, and activation of Rab5c by EGFR signaling was necessary to promote the intracellular association of AMAP1 and PRKD2. Our results suggest a novel mechanism by which EGFR signaling promotes the invasiveness of some breast cancer cells via integrin recycling.     

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer

5''-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.     

EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.     

Downmodulation of TGF-α protein expression with antisense oligonucleotides inhibits proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells

Interruption of an autocrine growth pathway involving TGF-α and EGFR may inhibit tumor growth and improve survival in head and neck cancer patients. We previously demonstrated that biopsy specimens and established cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress TGF-α and its receptor, epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies showed that TGF-α and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting a role for this ligand-receptor pair in an autocrine growth pathway. To confirm that TGF-α contributes to autocrine growth, we examined the effect of down regulation of TGF-α protein on SCCHN cell proliferation. Treatment of 6 SCCHN cell lines with antisense oligodeoxynucleotides targeting the translation start site of human TGF-α mRNA decreased TGF-α protein production by up to 93% and reduced cell proliferation by a mean of 76.2% compared to a 9.7% reduction with sense oligonucleotide (range P<0R > = 0.036–0.0001). TGF-α antisense oligonucleotide exposure also decreased TGF-α protein levels in normal oropharyngeal mucosal epithelial cells, however their growth rate was not affected. These findings indicate that TGF-α is participating in an autocrine signaling pathway in transformed, but not in normal mucosal epithelial cells, that promotes proliferation. J. Cell. Biochem. 69:55–62, 1998. © 1998 Wiley-Liss, Inc.