Synergistic Effects of Neratinib in Combination With Palbociclib or Miransertib in Brain Cancer Cells.

Background: Aberrant expression and activation of epidermal growth factor receptor (EGFR) resulted in approval of several forms of EGFR inhibitors in the treatment of patients with a wide range of epithelial cancers. However, no EGFR inhibitor has yet been approved for the treatment of patients with brain cancer, indicating that targeting EGFR alone may not be sufficient in some patients. Methods: In this study, we investigated the role of all members of the EGFR family, other growth factor receptors, cell-cycle proteins, and downstream cell signaling pathways (e.g., mitogen-activated protein kinase (MAPK), serine/threonine protein kinase (AKT), signal transducer and activator of transcription (STAT3), Src, Abelson murine leukemia viral oncogene homolog (Abl)) on the growth of a panel of human brain cancer cell lines (HBCCLs). We examined the growth response of HBCCLs to treatment with 17 targeted agents compared to two cytotoxic drugs. Results: Of the targeted agents, the irreversible pan-human epidermal growth factor receptor (HER) inhibitors neratinib and afatinib were more effective than erlotinib and lapatinib at inhibiting the growth of all HBCCLs, and the cyclin-dependent kinase (CDK)1/2/5/9 inhibitor dinaciclib was the most potent targeted agent. We found that treatment with Src/Abl/c-kit inhibitor dasatinib, signal transducer and activator of transcription (STAT3) inhibitor stattic, Abl/platelet-derived growth factor receptor (PDGFR)α/vascular endothelial growth factor (VEGFR)2/fibroblast growth factor receptor (FGFR)1 inhibitor ponatinib, and the tropomyosin receptor kinase (TRK)/ROS proto-oncogene 1 receptor tyrosine kinase (ROS)/anaplastic lymphoma kinase (ALK) inhibitor entrectinib, also inhibited the growth of all HBCCLs. Interestingly, these agents were more effective in inhibiting growth of HBCCLs when proliferating at a slower rate. In addition to inhibiting the proliferation of HBCCLs, treatment with neratinib, dinaciclib, dasatinib, stattic and trametinib inhibited the migration of brain tumor cell line A172. Conclusions: Notably, we found that treatment with neratinib in combination with palbociclib (CDK4/6 inhibitor), or miransertib (AKT1/2/3 inhibitor) resulted in synergistic growth inhibition of all HBCCLs. Our results support that repurposing drugs like neratinib in combination with the palbociclib or miransertib may be of therapeutic potential in brain cancer and warrants further investigations.

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells.

Background: Of various human epidermal growth factor receptor (HER) inhibitors, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan (T-Dxd) has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients, with tumor heterogeneity being one contributing factor. Methods: We investigated the effect of various types of targeted agents on growth in vitro and migration of a panel of human stomach cancer cells (HSCCLs) and the impact of cell proliferation rate on the anti-tumor activities of these agents. We also investigated the association between the cell surface expression of the HER family members, hepatocyte growth factor receptor (c-Met), anaplastic lymphoma kinase (ALK)7 and cancer stem cell markers CD44 and CD133, and the response to the targeted agents. Results: Of the 18 agents examined, the cyclin dependent kinase (CDK) 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at 50% inhibitory concentration (IC50) values between 9 nM to 23 nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor (TKI) lapatinib and the EGFR-specific TKI erlotinib in inhibiting the growth of HSCCLs. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs, with the IC50 values ranging from 2 nM to 7 µM. Many of these agents were more effective in inhibiting the growth of HSCCLs when they were proliferating at a slower rate. Treatment with neratinib, afatinib, dinaciclib, dasatinib, stattic, miransertib and paclitaxel significantly inhibited migration of stomach cancer cells. Interestingly, treatment with a combination of afatinib and dasatinib or afatinib and miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. Conclusions: These results suggest that treatment with a combination of these agents may be of therapeutic value in stomach cancer and warrants further investigations.

HER2 Expression Is Predictive of Survival in Cetuximab Treated Patients with RAS Wild Type Metastatic Colorectal Cancer.

The overexpressed HER2 is an important target for treatment with monoclonal antibody (mAb) trastuzumab, only in patients with breast and gastric cancers, and is an emerging therapeutic biomarker in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) mAbs cetuximab and panitumumab. In this study, we investigated the relative expression and predictive value of all human epidermal growth factor receptor (HER) family members in 144 cetuximab-treated patients with wild type RAS mCRC. The relative expression of EGFR and HER2 have also been examined in 21-paired primary tumours and their metastatic sites by immunohistochemistry. Of the 144 cases examined, 25%, 97%, 79%, 48%, and 10% were positive for EGFR, HER2, HER3, and HER4 and all four HER family members, respectively. The expression of EGFR was an indicator of poorer overall survival and the membranous expression of HER2 and HER3 3+ intensity was associated with a shorter progression free survival (PFS). In contrast, the cytoplasmic expression of HER2 was associated with better PFS. In 48% and 71% of the cases, there were discordance in the expression of EGFR or one or more HER family members in paired primary and related metastatic tumours, respectively. Our results implicate the importance of a large prospective investigation of the expression level and predictive value of not only the therapeutic target (i.e., EGFR protein) but also HER2 and other HER family members as therapeutic targets, or for response to therapy with anti-EGFR mAbs and other forms of HER inhibitors, in both the primary tumours and metastatic sites in mCRC.

Synergistic activity of agents targeting growth factor receptors, CDKs and downstream signaling molecules in a panel of pancreatic cancer cell lines and the identification of antagonistic combinations: Implications for future clinical trials in pancreatic cancer.

Pancreatic cancer is one of the most aggressive, heterogeneous and fatal type of human cancers for which more effective therapeutic agents are urgently needed. Here, we investigated the sensitivity of a panel of seven human pancreatic cancer cell lines (HPCCLs) to treatment with various tyrosine kinase inhibitors (TKIs), cyclin­dependent kinase (CDK) inhibitors, an inhibitor of STAT3 stattic, and a cytotoxic agent gemcitabine both as single agents and in combination. The membranous expression of various receptors and the effect of selected agents on cell cycle distribution, cell signaling pathways and migration was determined using flow cytometry, western blot analysis and scratch wound healing assays, respectively. While the expression of both HER­3 and HER­4 was low or negative, the expression of EGFR and HER2 was high or intermediate in all HPCCLs. Of all the agents examined, the CDK1/2/5/9 inhibitor, dinacicilib, was the most potent agent which inhibited the proliferation of all seven HPCCLs with IC50 values of ≤10 nM, followed by SRC targeting TKI dasatinib (IC50 of ≤258 nM), gemcitabine (IC50 of ≤330 nM), stattic (IC50 of ≤2 µM) and the irreversible pan­HER TKI afatinib (IC50 of ≤2.95 µM). Treatment with afatinib and dasatinib inhibited the ligand­induced phosphorylation of EGFR and SRC respectively. Statistically significant associations were found between HER2 expression and response to treatment with the ALK/IGF­IR/InsR inhibitor ceritinib and fibroblast growth factor receptor (FGFR)1/2/3 inhibitor AZD4547, HER3 and IGF­IR expression and their response to treatment with TKIs targeting HER family members (erlotinib and afatinib), and c­MET and ALK7 expression and their response to treatment with stattic. Interestingly, treatment with a combination of afatinib with dasatinib and gemcitabine with dasatinib resulted in synergistic tumor growth inhibition in all HPCCLs examined. In contrast, the combination of afatinib with dinaciclib was found to be antagonistic. Finally, the treatment with afatinib, dasatinib and dinaciclib strongly inhibited the migration of all HPCCLs examined. In conclusion, the CDK1/2/5/9 inhibitor dinaciclib, irreversible pan­HER TKI afatinib and SRC targeting TKI dasatinib were most effective at inhibiting the proliferation and migration of HPCCLs and the combination of afatinib with dasatinib and gemcitabine with dasatinib led to synergistic tumor growth inhibition in all HPCCLs examined. Our results support further investigation on the therapeutic potential of these combinations in future clinical trials in pancreatic cancer.

Co-expression and prognostic significance of putative CSC markers CD44, CD133, wild-type EGFR and EGFRvIII in metastatic colorectal cancer.

The presence of colorectal cancer stem cells (CSCs) have been associated with tumour initiation and resistance to therapy. This study investigated the co-expression and prognostic significance of the CSCs biomarkers CD44 and CD133 with wild-type EGFR (wtEGFR) and EGFRvIII in colorectal cancer (CRC). The expression of these biomarkers were determined in tumours from 70 patients with metastatic CRC by immunohistochemistry, and in a panel of human CRC cell lines, and their variants with acquired-resistance to EGFR inhibitors, by flow cytometry. The expression of CD44, CD133, wtEGFR and EGFRvIII were present in 17%, 23%, 26% and 13% of cases and the co-expression of CD44/CD133 with wtEGFR and EGFRvIII were present in 9% and 3% of the cases respectively. Only co-expression of CSCs/EGFRvIII (P = 0.037), and amphiregulin (P = 0.017) were associated with worse overall survival. Interestingly, disease-free survival was improved in BTC expressing patients (P = 0.025). In vitro CD133 expression and its co-expression with CD44 were associated with primary-resistance to irinotecan and acquired-resistance to anti-EGFR inhibitors respectively. Our results suggest co-expression of CSCs and EGFRvIII could be potential biomarkers of worse overall survival and resistance to therapy in patients with mCRC and warrants further validation in a larger cohort.

Co-expression and prognostic significance of the HER family members, EGFRvIII, c-MET, CD44 in patients with ovarian cancer.

EGFR and HER-2 are important targets but none of the monoclonal antibodies or small molecule tyrosine kinase inhibitors specific for the HER members has been approved for the treatment of patients with ovarian cancers. In some studies, co-expression of other growth factor receptors has been associated with resistance to therapy with the HER inhibitors. The aim of the present study was to determine the relative expression, cellular location, and prognostic significance of HER-family members, the EGFR mutant (EGFRvIII) c-MET, IGF-1R and the cancer stem cell biomarker CD44 in 60 patients with FIGO stage III and IV ovarian cancer. At cut off >5% of tumour cells with positive staining, 62%, 59%, 65% and 45% of the cases were EGFR, HER-2, HER-3 and HER-4 positive, and 3%, 22% and 48.3% of the cases were positive for EGFRvIII, c-MET, and CD44 respectively. Interestingly, 23% co-expressed all four members of the HER family. On univariate analysis, only EGFR staining at >50% of tumour cells (HR = 3.57, p = 0.038) and CD44 staining at 3+ intensity (HR = 7.99, p = 0.004) were associated with a poorer overall survival. EGFR expression (HR = 2.83, p = 0.019) and its co-expression with HER-2, HER-3, HER-2/HER-3, and c-MET were all associated with poorer disease-free survival. Our results suggest co-expression of the HER-family members is common in Stage III and IV ovarian cancer patients. Further studies on the prognostic significance and predictive value of all HER family member proteins for the response to treatment with various forms of the HER inhibitors are warranted.

The impact of co-expression of wild-type EGFR and its ligands determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer.

Anti-EGFR mAbs cetuximab and panitumumab are routinely used for the treatment of patients with KRAS-wild type metastatic colorectal cancer (mCRC). However, in some patients their efficacy remains modest and with no clear association between the EGFR protein expression determined by PharmDx™ kit, and response to anti-EGFR therapies. Therefore, we investigated the relative expression and predictive value of wild-type EGFR (wtEGFR), mutated EGFRvIII and EGFR ligand proteins in mCRC patients treated with cetuximab. The expression levels of wtEGFR, EGFRvIII, and EGFR ligand were determined by immunohistochemistry (IHC) in 60 tumour specimens using specific antibodies. Sections were scored according to the percentage of positive tumour cells, intensity and cellular location of staining, and these were associated with response, overall survival (OS) and progression-free survival (PFS). At cut-off value > 5%, wtEGFR, and EGFRvIII were present in 44%, and 41%, betacellulin (BTC) in 72%, followed by epigen (67%), TGFα (58%), amphiregulin (34%), EGF (31%) of the cases, respectively and 96% of the wtEGFR positive cases had co-expression of at least one ligand. We found a significant association between the expression of wtEGFR and poor response to cetuximab. In addition, the co-expression of wtEGFR with one ligand at a cut-off value of > 5% and > 10% was associated with worse response to cetuximab (P = 0.021, and P = 0.005 respectively). We found a 3-fold and 5-fold increased risk of shorter OS with expression of BTC and epigen. Interestingly, the expression of wtEGFR and its co-expression with one or two ligands was associated with shorter PFS but not with OS. The relative expression of wtEGFR and its competing ligands, which is the target for therapeutic interventions with anti-EGFR antibodies, could serve as a more reliable predictive biomarker of response to therapy with anti-EGFR mAbs in mCRC patients and warrants further investigation in large prospective studies.

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers.

BACKGROUND: The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment. METHODS: In this study, we investigated the molecular mechanisms underlying development of acquired resistance in DiFi colorectal cancer cells to the anti-EGFR mAb ICR62 (termed DiFi62) and to the small molecule tyrosine kinase inhibitor (TKI) gefitinib (termed DiFiG) using a range of techniques. RESULTS: Compared with the findings from parental DiFi and DiFiG cells, development of acquired resistance to anti-EGFR mAb ICR62 in DiFi62 cells was accompanied by an increase in cell surface EGFR and increased phosphorylation of HER-2 and HER-3. Interestingly, DiFi62 cells also acquired resistance to treatment with anti-EGFR mAbs cetuximab and ICR61, which bind to other distinct epitopes on the extracellular domain of EGFR, but these cells remained equally sensitive as the parental cells to treatment with pan-HER inhibitors such as afatinib. CONCLUSIONS: Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed.

Co-expression of HER family members in patients with Dukes' C and D colon cancer and their impacts on patient prognosis and survival.

The human epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with metastatic colorectal cancer and anti-EGFR antibodies cetuximab and panitumumab have been approved for the treatment of such patients. Despite these advances, the duration of response in some patients can be limited. Since, EGFR is capable of forming heterodimers with the other members of the HER (Human epidermal receptor) family, it is important to investigate the co-expression and prognostic significance of all members of the HER family in colorectal cancer patients. The expression of the HER family members were determined in tumour specimens from 86 patients with Dukes' C and D (metastatic) colon cancer using immunohistochemistry. Sections were scored by the percentage of positive tumour cells and intensity of staining. Their associations with clinicopathological parameters, and overall survival and disease free survival were evaluated using univariate and multivariate analysis. Overall, 43%, 77%, 52% and 92% of the cases were EGFR, HER-2, HER-3 and HER-4 positive respectively. Interestingly, 35%, 24%, 43%, and 18% of the cases had co-expression of EGFR/HER-2, EGFR/HER-3, EGFR/HER-4 and all four members of the HER family respectively. Of these, only the expression of EGFR and co-expression of EGFR/HER-4 were associated with poorer disease-free survival in both univariate and multivariate analysis. Co-expression of all members of the HER family in colon cancer supports the need for further investigations on their predictive value for response to therapy with anti-EGFR mAbs and whether such sub-population of patients may benefit from therapy with the new generation of pan-HER inhibitors.

Prognostic significance and targeting of HER family in colorectal cancer.

Colorectal cancer is one of the leading causes of cancer deaths. At present, anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) cetuximab and panitumumab and anti-vascular endothelial growth factor (VEGF) mAb bevacizumab have been incorporated into treatment paradigms for patients with refractory metastatic colorectal cancer. However, many patients simply do not respond to these treatments or eventually acquire resistance following a short course therapy. In this article, we review the literature for studies on the expression patterns, prognostic significance and predictive value of HER (also called erbB) family members and other factors for response to therapy with the HER inhibitors in colorectal cancer. We discuss some of the advances, challenges as well as future opportunities for more effective targeting of the HER receptors using a cocktail of HER inhibitors (e.g. dual and pan HER TKIs, monospecific or bispecific antibodies) in combination with other therapeutic interventions.

Growth response of human colorectal tumour cell lines to treatment with afatinib (BIBW2992), an irreversible erbB family blocker, and its association with expression of HER family members.

Despite the approval of the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs), cetuximab and panitumumab, for the treatment of colorectal cancer patients, there is currently no reliable predictive marker for response to therapy. In addition, the duration of response is often limited. Therefore, this study aimed to investigate the effect of afatinib, an irreversible erbB family blocker, as a single agent or in combination with cytotoxic drugs (5-fluorouracil, irinotecan and oxaliplatin) or mAb ICR62 on the proliferation of a panel of human colorectal tumour cell lines and the association between the expression levels of the EGFR family members and response to treatment. Of the cells examined, EGFR-overexpressing DiFi cells were the most sensitive to treatment with both afatinib (IC50=45 nM) and ICR62 (IC50=4.33 nM). Afatinib also inhibited the growth of other tumour cell lines with IC50 values which ranged from 0.33 µM (CCL-221) to 1.62 µM (HCT-116). A significant association was found between the co-expression of EGFR, human epidermal growth factor receptor (HER)-2 and HER-3 and response to treatment with afatinib (R=0.915, P=0.021). Treat-ment with afatinib and cytotoxic drugs was accompanied by an increase in the proportion of these cells in the sub-G0/G1 and in the S and G2/M phase of the cell cycle, respectively. We conclude that afatinib as monotherapy or in combination with other drugs shows activity in colorectal tumour cells and that determination of the co-expression of HER family members should be conducted in clinical trials using drugs targeting erbB signaling. This approach could lead to the identification of a specific subpopulation of cancer patients more likely to benefit from erbB-directed therapy.