Amplification of ERBB2 or increased heregulin concentrations lead to extracellular signal-regulated kinase 1/2 signaling.
Scientists have implicated ERBB2 signaling as the cause of de novo and acquired resistance to the anticancer antibody cetuximab (marketed as Erbitux). In vitro and in vivo studies showed that either amplification of the ERBB2 gene itself or heregulin upregulation leads to extracellular signal-regulated kinase 1/2 signaling and resistance to the EGFR-targeting antibody even in the absence of other resistance-related gene mutations.
The international team led by researchers at the Dana-Farber Cancer Institute and Japan’s Kinki University School of Medicine also showed that inhibiting ERBB2 or disrupting ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vivo. Reporting their findings in Science Translational Medicine, Kinki University’s Kazuhiko Nakagawa, Ph.D., and Dana-Farber’s Pasi A. Jänne, Ph.D., confirmed the presence of ERBB2 amplification or high levels of circulating heregulin in a subset of colorectal cancer (CRC) patients with de novo or acquired resistance to cetuximab-based therapy.
The researchers suggest that combining ERBB2 inhibitors with cetuximab should be tested as an approach to treating patients with cetuximab-resistant tumors. Their published paper is titled “Activation of ERBB2 Signaling Causes Resistance to the EGFR-Directed Therapeutic Antibody Cetuximab.”
Mutations in genes such as KRAS, BRAF, and NRAS that cause aberrant activation of signaling pathways downstream of EGFR have been shown to result in de novo resistance to cetuximab therapy, the researchers explain. As a result, most of the clinical benefits of the drug are limited to patients with cancers that don’t harbor these oncogenic mutations. However, even among theoretically susceptible patients, cetuximab is not always clinically effective, suggesting other mechanisms are involved in de novo resistance. Moreover, the benefits of cetuximab in patients who do initially respond to therapy are hindered further because even these patients always ultimately develop resistance, the team adds.
They first generated cetuximab-resistant clones of the lung cancer-derived HCC827 cell line using a previously described method. In these cells, unlike in the parental HCC827 cells, cetuximab did not fully inhibit phospho-ERK1/2 (pERK1/2). The team carried out genome-wide copy number analysis to compare the genomes of the antibody-resistant cells with those of parental HCC827 cells.
The results showed that the cetuximab-resistant clones exhibited a large region of copy number gain on chromosome 17 encompassing the ERBB2 oncogene. Amplification of ERBB2 was confirmed using FISH analysis, and the researchers also found that the cetuximab-resistant HCC827 cells expressed higher levels of both total ERBB2 and phospho-ERBB2 than parental cells.
To confirm these findings in another cell line they generated cetuximab-resistant clones of the GEO CRC cell line and again found that a number of the resulting clones harbored ERBB2 gene amplifications and increased levels of ERBB2. As in the cetuximab-resistant HCC827 clones, treatment of the resistant GEO CRC cells using the cetuximab didn’t effectively downregulate pERK1/2.
To determine whether ERBB2 plays a causal role in cetuximab resistance, the team depleted ERBB2 in HCC827 cetuximab-resistant cells using an ERBB2-specific shRNA. The result was restoration of both cetuximab sensitivity and its ability to down-regulate pERK1/2.
Testing different combinations of drugs on one of the cetuximab-resistant HCC827 clones and one of the cetuximab-resistant CRC clones, the team also found that a combination of the ERBB2-directed antibody trastuzumab in combination with cetuximab was capable of blocking cell growth. Treating cetuximab-resistant HCC827 cells with the ERBB2 kinase inhibitor lapatinib also restored sensitivity to cetuximab and the antibody’s ability to inhibit pERK1/2.
“These findings suggest that ERBB2 amplification is the principal mechanism of resistance to cetuximab in both NSCLC and CRC cells and that inhibition of ERBB2, in conjunction with cetuximab, represents a potential treatment strategy for patients with acquired cetuximab resistance,” the authors state.
To evaluate whether ERBB2 could confer resistance in other cancer cell lines, the team introduced the ERBB2 gene to the normally cetuximab-sensitive HNSCC cell line HN11 and the similarly cetuximab-sensitive NSCLC line H1648. In both cases Introduction of ERBB2 rendered the cells resistant to cetuximab, and the antibody was unable to downregulate pERK1/2 in HCC827 and HN11 cells that overexpressed the ERBB2 protein.
The next step was to see whether activation of ERK1/2 signaling was enough to make cells resistant to cetuximab, in the absence of ERBB2 overexpression. To this end the researchers introduced BRAF V600E into HCC827 or HN11 cells and evaluated the effects of cetuximab. BRAF V600E is associated with cetuximab resistance in preclinical models and in CRC patients. In both the resulting cell lines the antibody no longer fully inhibited pERK1/2, and the cells became resistant.
In further support of the notion that ERK1/2 signaling is the critical factor in ERBB2-related resistance, the team found that ERBB2 did not inhibit cetuximab binding to EGFR in either cetuximab-resistant HCC827 cells or in HN11 ERBB2 cells nor did ERBB2 interfere with cetuximab-mediated internalization of EGFR. “Collectively, these findings suggest that the principal mechanism by which ERBB2 causes cetuximab resistance is by activating ERK1/2 signaling,” they write.
The team then looked at a cetuximab-resistant version of a different cell line, A431, which expresses increased levels of pERBB2 and pERBB3 but doesn’t show any evidence of ERBB2 amplification or increased ERBB2 levels. These A431CR cells were found to produce about 2.5 times more heregulin in cell culture than the parental A431 cells.
Previous work has shown that in the presence of heregulin, ERBB3 dimerizes with ERBB2 and both proteins are phosphorylated. So to investigate what effect heregulin loss had on cetuximab sensitivity, the researchers depleted heregulin in the cetuximab-resistant A431 cells using siRNAs. As well as resulting in lower phosphorylation of ERBB3 and AKT (which is a known mediator of ERBB3 signaling), the cells demonstrated greater sensitivity to cetuximab, and the antibody readily reduced pERK1/2. Conversely, adding heregulin to different cetuximab-sensitive cancer cell lines resulted in dose-dependent decreases in their sensitivity to the antibody, and resulted in ERBB2 and ERBB3 phosphorylation.
The next stage was to see whether inhibiting ERBB2 could have any beneficial therapeutic effects. In vitro tests showed that ERBB2 silencing using an ERBB2-specific siRNA resulted in increased sensitivity of cell lines to to cetuximab.
Interestingly, both A431 and cetuximab-resistant A431 cells were sensitive to the EGFR/ERBB2 dual kinase inhibitor lapatinib. The resistant cells were also sensitive to cetuximab combined with pertuzumab, an antibody that disrupts ERBB2/ERBB3 dimerization. Immunoblotting assays showed that in A431CR cells cetuximab was able to down-regulate pERK1/2 in the presence of pertuzumab, while ERK1/2 remained phosphorylated in the absence of pertuzumab.
The team then tested different EGFR-targeting antibodies in vivo, including the EGFR kinase inhibitor gefitinib, and cetuximab. Both effectively inhibited xenografts generated from GFP-tagged HCC827 cells, whereas only gefitinib inhibited the growth of HCC827 ERBB2 xenografts, which were resistant to cetuximab.
Cetuximab treatment led to inhibition of pEGFR and down-regulation of total EGFR in the GFP-tagged HCC827 tumors in mice, but not in animals in HCC827 ERBB2 tumors. Coprecipitation studies suggested the formation of EGFR/ERBB2 heterodimers in cetuximab-resistant HCC827 ERBB2 tumors. Cetuximab either alone or in combination with pertuzumab effectively inhibited the growth of A431 xenografts, but only the combination of both antibodies regressed cexuximab-resistant A431 tumors in vivo.
To try and extrapolate their in vitro and in vivo findings to a clinical setting, the researchers evaluated tumor and blood specimens from cetuximab-treated patients who demonstrated either de novo or acquired resistance to the antibody.
They identified 13 with de novo ERBB2 amplifications and 220 patients without de novo ERBB2 amplification, who had been treated with cetuximab alone or in combination with chemotherapy. Median progression-free survival (PFS) was longer (149 days) among patients without ERBB2 amplification compared with those whose tumors did carry amplifications in the ERBB2 gene (89 days). Median overall survival was also longer for patients with nonamplified ERBB2 tumors (515 days) than for those with ERBB2 amplifications (307 days). The findings held true even when only patients with KRAS wild-type tumors were evaluated.
The researchers then analyzed tumor specimens taken before and after cetuximab treatment, from two CRC patients who developed clinical cetuximab resistance. In both cases there were substantially more ERBB2-amplified tumor cells in posttreatment tumors than in pretreatment tumors.
Circulating serum levels of the ERBB2/HER2 extracellular domain were then measured in another nine patients. Two patients who had previously had a partial response to cetuximab-based therapy demonstrated much higher serum HER2 ECD levels at the time of disease progression than before treatment.
The team subsequently looked at the relationship between heregulin and de novo cetuximab resistance in 70 cetuximab-treated CRC patients. Although plasma heregulin concentrations varied widely, they were consistently lower in patients who had a partial response (PR) to cetuximab-based therapy than in those who had either stable disease (SD) or progressive disease, even among patients specifically with KRAS wild-type cancers.
They separately isolated RNA from pretreatment tumors from 44 of the 70 CRC patients and correlated heregulin concentration with cetuximab efficacy. This confirmed that patients achieving a PR had significantly lower levels of tumor heregulin expression compared with those with SD or PD, irrespective of KRAS status. The group of patients with low heregulin expression in addition had significantly longer PFS and overall survival than the cohort with high heregulin expression when treated with cetuximab.
The results were similar in patients who acquired cetuximab resistance. Serum heregulin levels were evaluated in seven patients who initially achieved a PR with cetuximab-based therapy but subsequently developed resistance. For each individual, plasma heregulin concentrations were significantly higher after cetuximab therapy than before the treatment was started.
“Collectively, these clinical studies further support our in vitro and in vivo studies and demonstrate that both ERBB2 amplification and increased heregulin levels are associated with both de novo and acquired resistance to cetuximab-based therapy in CRC patient,” the authors conclude. “ERRB2 amplification is a unique mechanism of drug resistance in the case of cetuximab because ERBB2-amplified, cetuximab-resistant NSCLC cells remain sensitive to the EGFR kinase inhibitor gefitinib in vitro and in vivo. Our findings are directly relevant to patients who develop acquired resistance to cetuximab-based therapy and may help guide subsequent treatment.”
They further suggest that prospective clinical trials of cetuximab should evaluate drug resistance mechanisms including ERBB2 amplification and heregulin measurements at the time of disease progression. “For patients with evidence of one of these drug resistance mechanisms, cetuximab combined with ERBB2-targeted therapy (for both mechanisms) or with an anti-ERBB3 antibody (heregulin only) should be further evaluated in clinical trials.”