Background
Colorectal cancer represents the third most common cancer and the second leading cause of cancer-related deaths in the United States and Western Europe [
1,
2]. However, despite implementation of multimodal treatment approaches and novel targeted therapeutics within the last two decades [
3,
4], the occurrence of distant metastases still limits the prognosis of affected patients. In this context, up to 50% of patients with CRC develop metastatic disease recurrence, predominantly in the liver and the lung, and, until now, surgical resection represents the only curative strategy [
5‐
7]. Unfortunately, resectability is technically not always feasible, and disease recurrence after metastasis resection is frequently observed [
8,
9]. Consequently, there is an urgent clinical need to develop novel agents and treatment strategies to inhibit metastatic cancer progression.
In metastatic CRC, treatment regimens were commonly based on 5-fluorouracil (5-FU) and, recently, in combination with irinotecan or oxaliplatin [
10]. Due to the lack of specificity of these drugs, there have been major initiatives in targeted-therapy approaches. A primary focus was EGF receptor signaling, which plays a key role in CRC development and progression [
11‐
13]. Major clinical trials, including recent data from the CELIM study, have demonstrated that initially unresectable CRC liver metastases can be surgically removed after combined EGFR inhibition and chemotherapy (CTx), resulting in a better survival of these patients [
14,
15]. Unfortunately, most CRC develop resistance against EGFR-targeting agents, which ultimately limits this therapeutic strategy [
16,
17]. Therefore, the evaluation of alternative therapeutic targets is crucial for the implementation of innovative treatment approaches. In this context, the transmembrane receptors HER-2 and HER-3 represent interesting candidates.
HER-2, a member of the EGF receptor family of receptor tyrosine kinases (Erb), commonly referred as ErbB2, represents a prognostic biomarker in breast cancer and has been a molecular target for many years [
18,
19]. Recently, HER-2 inhibition has also been integrated into therapeutic strategies for metastatic gastric cancer [
20,
21]. Among other studies, the ToGA-trial demonstrated HER-2 positivity in about 20–30% of adenocarcinomas of the stomach and gastro-esophageal junction [
22], and a survival benefit upon treatment with trastuzumab using a specifically modified immunohistochemistry (IHC) scoring algorithm, which differed from breast cancer [
20]. While data about the prognostic and functional relevance of HER-2 expression are still limited for most gastrointestinal malignancies [
21,
23], we have recently reported HER-2 positivity in more than 20% of primary rectal cancer [
24], and overexpression of HER-2 in nearly 10% of CRC-derived liver metastases [
25]. Moreover, we observed overexpression of another member of the EGF receptor family, HER-3, in approximately 70% of CRC-derived liver metastases [
25]. This observation is of high clinical interest because novel HER-3 inhibitors have been recently developed and are currently being tested within early phase clinical trials [
26,
27].
In the present study, we determined the protein expression of HER-2 and HER-3 in 12 CRC cell lines using immunocytochemistry (ICC). Selected cell lines were treated with the HER-2-specific antibodies trastuzumab or pertuzumab, which either prevent ligand binding or dimerization of HER-2 with other HER receptors. Additionally, cells were incubated with the antibody-drug conjugate T-DM1, the dual tyrosine kinase inhibitor lapatinib, and the irreversible Pan-ErbB (HER-1/HER-2/HER-4) inhibitor afatinib. Specific targeting of Erb receptors was combined with 5-FU and oxaliplatin, which represents a standard regime in the clinical setting. Finally, we evaluated the frequency of HER-3 protein expression in patients with primary rectal cancer using IHC.
Methods
Cell lines and cell culture
Human CRC cell lines HT29, SW403, SW837, SW1116, LS513, LS1034, Caco-2, SW1463, SW480, SW620, HCT116, and LS411N were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in their recommended media (Invitrogen, Karlsruhe, Germany), supplemented with 2 mM L-glutamine (Lonza, Verviers, Belgium) and 10% fetal bovine serum (Biochrome, Berlin, Germany). Periodically, mycoplasma contamination was excluded using the MycoAlert® Mycoplasma Detection Kit (Lonza, Cologne, Germany), and cell-line cross-contamination was excluded using short tandem repeat profiling [
28]. Relevant characteristics of these cell lines are summarized in Table
1.
Table 1
Genetic characteristics and HER-2/HER-3 immunostaining of 12 CRC cell lines
HT29 | + | - | + | MSS | - | 1+ | 3+ |
SW403 | + | + | + | MSS | - | 1+ | 1+ |
SW837 | + | + | + | MSS | - | 2+ | 1+ |
SW1116 | + | + | + | MSS | - | 2+ | 3+ |
LS513 | - | + | - | MSS | - | 2+ | 3+ |
LS1034 | + | + | + | MSS | - | 2+ | 3+ |
Caco-2 | + | - | + | MSS | - | 2+ | 1+ |
SW1463 | + | + | + | MSS | - | 1+ | 3+ |
SW480 | + | + | + | MSS | + | 0 | 0 |
SW620 | + | + | + | MSS | - | 0 | 0 |
HCT116 | - | + | - | MSS | - | 0 | 0 |
LS411N | + | - | + | MSS | + | 2+ | 1+ |
Drugs
Trastuzumab, pertuzumab, and T-DM1 (Roche, Penzberg, Germany) were obtained by the local pharmacy of the University Medical Center in Goettingen. Small-molecule inhibitors afatinib and lapatinib were purchased from Santa Cruz (Dallas, TX), and 5-FU and oxaliplatin from Sigma (Munich, Germany).
Cellular viability assays
Cellular viability was determined using the CellTiter-Blue® reagent (Promega, Madison, WI), as previously described [
29]. Briefly, cell lines growing in log-phase were seeded at different densities (8000 cells per well for LS513; 6000 cells for LS1034; and 6000 cells for SW837, respectively) into black clear bottom 96-well plates (Corning, Corning, NY). Cells were allowed to adhere overnight, and drugs were added with increasing concentrations. Twenty-four, 48, and 72 h upon treatment start, reduction of resazurin to resorufin was measured using a plate reader (VICTOR™ X4, Perkin Elmer, Waltham, MA) according to the manufacturer’s instructions. Cellular viability of antibody-treated cells was compared to untreated cells, and viability of inhibitor-treated cells was compared to DMSO-controls, as previously described [
30]. All experiments were performed as three independent replicates, with three technical replicates per plate.
Western blot analysis
Cell lines were seeded into six-well plates (106 cells per well) with increasing concentrations for the indicated drugs. Twenty-four hours later, cells were stimulated with 100 ng/ml neuregulin (NRG, Cell Signaling, Danvers, MA) for 10 min at 37 °C. Subsequently, cells were lysed using RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% Na-deoxycholate, 1% NP-40, 2 mM EDTA) followed by sonification. Finally, 20 μg of whole-cell protein lysate was resolved on a 10% Bis-Tris gel (Roth, Karlsruhe, Germany) at 30 mA per gel. Proteins were transferred by wet blotting (Criterion™ blotter, Bio-Rad, Hercules, CA) onto a PVDF membrane (Merck-Millipore, Billerica, MA), and probed with primary antibodies p-Akt (1:1000; Cell Signaling), Akt (1:1000; Cell Signaling), and Actin (1:2000; Sigma, Saint Louis, MO) at 4 °C over night. On the next day, membranes were incubated for 2 h with the secondary antibody goat-anti-rabbit-HRP (1:30,000; Acris, Hiddenhausen, Germany). Signals were detected using ECL Luminata forte (Merck-Millipore) and a CCD camera system (LAS 4000mini; GE Healthcare, Munich, Germany).
Immunostaining
Cell lines with 70 to 80% confluence were trypsinized, washed with PBS, and fixed with buffered 4% formaldehyde (AppliChem, Darmstadt, Germany) over night at room temperature. Subsequently, cells were incubated with increasing concentrations of ethanol (60–100%) for 30 min, followed by incubation with isopropanol and xylene for 30 min. Finally cells were covered with hot paraffin for 10 min and embedded into a paraffin block.
HER-2 immunostaining was conducted using a PATHWAY® anti-HER-2/neu (4B5) rabbit monoclonal antibody (Ventana Medical Systems, Mannheim, Germany) on a Ventana BenchMark XT immunostainer (Ventana, Tucson, AZ), visualized by the ultraView Universal DAB Detection Kit (Ventana Medical Systems), as previously described [
24,
25]. HER-3 expression was determined in both cell lines and primary rectal cancer specimens using the anti c-erbB-3/HER-3 rabbit monoclonal antibody (clone SP71; Zytomed Systems, Berlin, Germany).
HER-2 and HER-3 scoring
For HER-2 scoring, we used an established protocol, which has been developed within the ToGA-trial and which is now being used to determine HER-2 protein expression in patients with adenocarcinoma of the stomach and the gastroesophageal junction [
20,
22,
31,
32]. Importantly, we have previously used this protocol to score HER-2 and HER-3 expression in primary rectal cancers and CRC liver metastases [
24,
25].
Cell lines or cancer cells from formalin-fixed paraffin embedded patient samples were considered ICC 2+ if at least 10% of the tumor cells had a medium membrane staining for HER-2 or HER-3, respectively, at high magnification (10x, 20x magnified), or ICC 3+ if at least 10% of the tumor cells had a strong membrane staining at low magnification (2.5x, 5x magnified). No membrane staining was scored ICC 0, and weak membrane staining in at least 10% of the tumor cells was defined as ICC 1+ (40x magnified).
Statistical analysis
Significant effects in cellular viability assays were analyzed using logistic regression with generalized linear models (glm) and analysis of variance (ANOVA). In the linear model, the cellular viability (in percent) was modeled as dependent on different replicates, different duration effects (24 h, 48 h, 72 h), a log10 dose effect and dose:duration interaction effects. Model comparisons were performed via ANOVA using the F-Test to assess whether the addition of the duration, dose or interaction variables adds significant information to the model. Statistical analyses were conducted using R statistical computing environment version 3.1.1. The (estimated) half maximal effective concentration (EC
50) was estimated based on the fitted logistic regression curves for each measurement series. If the EC
50 lies outside the range of measured doses, extrapolations can be inaccurate and lead to very large estimates. Comparisons of two measurement series were performed using a similar logistic regression model. Here, an additional drug combination effect plus all interaction effects were estimated. The ANOVA
P-value for the combination effect indicates that the drug combination displays a significantly different effect from the treatment with one drug alone (Additional file
5: Table S1).
The association of HER-3 expression levels with other clinico-pathological parameters was assessed using Fisher’s exact test. Survival rates were supplied by means of Kaplan-Meier analysis and tested using the Cox proportional hazards model. Time to recurrence (TTR) was defined as the interval between surgical resection of the primary tumor and disease recurrence, and cancer-specific survival (CSS) as time from surgical resection to Colorectal cancer-related death. The P-value was set to P < 0.05 to be considered statistically significant. Survival analysis was performed using the R package survival.
Discussion
Given its high incidence in the Western world, treatment of CRC remains an important interdisciplinary task. Although innovative surgical concepts and the implementation of multimodal treatment strategies have considerably improved both local control and oncological outcome [
38‐
40], systemic treatment of CRC patients with distant metastases remains a major clinical challenge. In this context, advances were obtained by combining cytostatic drugs such as 5-FU, oxaliplatin or irinotecan, and by the discovery and successful targeting of key signaling pathways, which promote colorectal carcinogenesis. Prime examples are the pharmacological inhibition of the vascular endothelial growth factor (VEGF) or the epidermal growth factor receptor (EGFR) in selected patients, which has become clinical routine [
37,
41].
However, and despite initial responses to these therapeutic approaches, secondary resistance frequently evolves over time, ultimately resulting in treatment failure [
16,
42]. The underlying mechanisms that lead to treatment resistance are quite complex and heterogeneous [
43]. Recent work demonstrated that prolonged inhibition of the EGF receptor (EGFR/ErbB1) leads to selection of Ras mutations as well as an increased expression of other members of the ErbB family, which can replace EGFR in EGF-mediated oncogenic signaling [
44,
45]. The two most prominent members of the ErbB family, which may substitute ErbB1 to escape EGFR inhibition, are HER-2 (ErbB2) and HER-3 (ErbB3). Especially the role of HER-2 has been described as a keystone in EGF-mediated growth activation in breast or gastric cancer [
19,
46]. Based on our present results and on previous analyses of primary rectal adenocarcinomas and CRC liver metastases, we found both HER-2 and HER-3 overexpressed in a substantial proportion of CRC [
24,
25], and in CRC cell lines. In addition, activating HER-2 mutations have been identified in CRC patients within the TCGA project as well as in several CRC cell lines [
11,
47]. More recently, activating HER-2 mutations were also detected in Lynch-like CRC [
48]. Consequently, both receptors represent attractive therapeutic targets.
Despite its activity in breast or gastric cancer [
19,
46], monotherapy with HER-2 inhibitors trastuzumab or pertuzumab only slightly reduced the viability of HER-2 positive CRC cells. Moreover, and in contrast to previous results in breast cancer [
49], the favorable effect of dual inhibition of HER-2 by simultaneous application of both antibodies showed only mediocre activity on CRC cell lines. A potential explanation why both antibodies, which specifically target the HER-2 receptor from outside the tumor cell, lack activity could be an intact intracellular tyrosine kinase activity resulting from heterodimerization with other ErbB family members or a constitutively active tyrosine kinase activity. Congruent with this assumption, inhibition of the tyrosine kinase activity of the HER-2 receptor by either lapatinib or afatinib dramatically impaired cellular viability in vitro. This effect was even more pronounced when treatment was combined with 5-FU and/or oxaliplatin, reflecting the clinically more relevant situation. Of note, the HERACLES phase-II trial recently tested as a proof of concept a combination of trastuzumab and lapatinib in patients with HER-2 positive metastatic CRC that were primarily resistant to cetuximab or panitumumab [
50]. This study demonstrated that approximately 5% of K-RAS exon 2 wild-type metastatic CRC are HER-2 positive, which is comparable to other malignancies with druggable molecular targets. Importantly, the treatment was well tolerated, and about 1/3 of the patients experienced either partial or complete response [
50].
The strongest impairment of cellular viability in our analyses, however, was observed upon treatment with the Pan-ErbB inhibitor afatinib, suggesting that other members of the ErbB family may be involved in EGF-mediated oncogenic signaling in colorectal cancer cells. These results together with the finding that HER-3 is expressed in a substantial proportion of CRC patients and CRC cell lines highlight the clinical rationale to simultaneously target members of the ErbB receptor family. Importantly, antibodies targeting HER-3 such as MM-121 (
ClinicalTrials.gov: NCT01451632), RG7116 (
ClinicalTrials.gov: NCT01482377) and U3–1287 are currently being tested in several clinical trials across various patient populations, including CRC patients. In cancers with ligand-dependent activation of HER-3, several studies suggest therapeutic potential of anti-HER-3 substances [
51]. Recently, HER-3 was identified as predictive factor for clinical outcome in K-RAS wild-type CRC patients treated with cetuximab [
52]. An ongoing clinical study evaluates treatment with MM-121 plus cetuximab versus MM-121 in combination with cetuximab plus irinotecan in CRC (
ClinicalTrials.gov: NCT01451632). Another multicenter study is recruiting participants to evaluate RG7116 alone, RG7116 in combination with cetuximab, or RG7116 plus erlotinib in patients with metastatic and/or locally advanced HER-3 positive solid tumors (
ClinicalTrials.gov: NCT01482377).
Conclusion
In conclusion, selective inhibition of the HER-2 receptor alone does not seem to represent a promising therapeutic strategy for CRC treatment, in contrast to breast cancer or other cancers of the gastro-intestinal tract. In clear contrast, simultaneous inhibition of different members of the ErbB receptor family dramatically abrogated cellular viability of CRC cells in vitro. Since both HER-2 and HER-3 are overexpressed in a relevant proportion of primary CRC and CRC liver metastases, targeting of HER-2 and HER-3 simultaneously may be considered as a potential therapeutic strategy in these patients upon failure of EGFR inhibition.
Acknowledgements
The authors are indebted to Jessica Eggert, Stefanie Mueller, Johanna Buschalsky, and Birgit Juenemann for excellent technical support. We also thank Dr. Markus Schirmer for critical discussions, Dr. Andreas Scheel for immunhistochemical analysis of HER-3 staining in patient samples and Prof. Dr. Michael Klintschar for short tandem repeat profiling analyses. Materials and data from this manuscript are part of the doctoral theses of Anna-Lena Metzger and Merle Kisly.
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