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Originally published as JCO Early Release 10.1200/JCO.2005.04.3489 on July 17 2006 © 2006 American Society of Clinical Oncology. Trastuzumab in Combination With Heregulin-Activated Her-2 (erbB-2) Triggers a Receptor-Enhanced Chemosensitivity Effect in the Absence of Her-2 Overexpression
From the Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston; Department of Medicine, Northwestern University Feinberg School of Medicine; Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL Address reprint requests to Ruth Lupu, PhD, Evanston Northwestern Healthcare Research Institute, 1001 University Place, Evanston, IL 60201; e-mail: r-lupu{at}northwestern.edu
Purpose The decision for treating breast cancer patients with trastuzumab is based on HER-2 amplification and/or overexpression. Methods Using MCF-7 cells (Her-2 ±) engineered to overexpress heregulin (MCF-7/HRG), a ligand for the Her-2/3/4 network, we investigated whether HRG-induced transactivation of Her-2 affected breast cancer cell sensitivity to chemotherapy and whether trastuzumab trigger receptor-enhanced chemosensitivity (REC) when combined with chemotherapy without Her-2 overexpression. Results MCF-7/HRG cells were more than 10-fold resistant to the alkylating agent cisplatin (CDDP), while trastuzumab coexposure completely reversed HRG-promoted CDDP resistance. A synergistic interaction between trastuzumab in combination with CDDP (paclitaxel or vincristine) was obtained in MCF-7/HRG cells. Trastuzumab prevented activation of the antiapoptotic and proliferative cascades and inhibited HRG-induced Her-2/3 phosphorylation. CDDP efficacy was enhanced by trastuzumab in cells expressing endogenously high levels of HRG. Conversely, trastuzumab coexposure was ineffective in enhancing chemotherapy efficacy in cells that did not secrete HRG, such as MCF-7 cells overexpressing a structural mutated HRG isoform. Therefore, trastuzumab-induced REC, in the absence of Her-2 overexpression, occurs through the kinase activity of Her-2/3. Interestingly, HRG expression in tumor biopsies from invasive breast carcinomas (n = 189) revealed that, whereas the minority (12%) of Her-2 positive tumors (n = 60; 32%) demonstrated Her-2 phosphorylation, the majority (67%) of HRG-overexpressing and Her-2 tumors (n = 57; 30%) were in active Her-2 status. Conclusion We demonstrate that assessment of HRG expression and Her-2 activation define a particular breast cancer patient population for which trastuzumab plus CDDP or taxol are extremely efficient without Her-2 overexpression.
The occurrence of HER-2 (erbB-2) gene amplification with overexpression occurs in approximately 25% of breast carcinomas and it is associated with a significantly lower overall survival rate and a shorter time to relapse.1-4 Her-2 overexpression plays a role in the response of breast cancer cells to lethal effects of chemotherapy.5-7 Earlier clinical studies suggested that Her-2 overexpression associates with improved outcome after anthracycline-based therapy as compared with alkylator-based therapy.8,9 Consistently, in vitro studies demonstrate that Her-2-driven signaling leads to increased sensitivity to doxorubicin, while Her-2 overexpression renders resistance to cisplatin (CDDP), a DNA-damaging agent.10-13 Trastuzumab (Herceptin; Genentech Inc, South San Francisco, CA), an antibody directed to the ectodomain of p185Her-2, is effective in the treatment of some Her-2–positive breast cancer patients.14-18 Trastuzumab has demonstrated activity as a single agent and when administered in conjunction with cytotoxic drugs.18,19 The use of trastuzumab in combination with CDDP in breast cancer culture cells,20-22 in xenografts,23 and in patients with Her-2-overexpressing metastatic breast cancer,24,25 results in tumoricidal effects higher than those achieved each as single agents. This synergistic interaction is not restricted to CDDP studies demonstrated that trastuzumab enhances the antitumor action of paclitaxel, docetaxel, vinorelbine, and cyclophosphamide.26-33 This effect, termed receptor-enhanced chemosensitivity (REC), has been suggested to specifically operate in Her-2–overexpressing cancer cells, but not in cells or tissues expressing physiological levels of the gene.31 Hence, the decision for treating breast cancer patients with trastuzumab either as single agent or in conjunction with cytotoxic drugs is based on HER-2 (erbB-2) gene amplification or overexpression. Several Her-2–positive cells and patients do not respond to trastuzumab.34,35 Although, it has been postulated that tumors exhibiting active Her-2 signaling might be most sensitive to trastuzumab,36-39 additional factors that could predict the effectivity of trastuzumab are still largely unknown. Her-2 is activated either by overexpression or by ligand-induced oligomerization with other erbB receptors from the epidermal growth factor (EGF) receptor family. Heregulin (HRG), a member of the EGF-like growth factor family, acts as a combinatorial ligand for Her-3 and Her-4 receptors.40-42 HRG binds Her-3 and Her-4 and activates the Her-2 receptor via Her-2 to Her-3 or Her-2 to Her-4 heterodimerization.41,42 We and others have presented evidence that HRG plays an important role in breast cancer. HRG is sufficient for the generation of adenocarcinomas while favoring the metastatic spread of breast cancer in vivo.43-46 Blockade of HRG expression inhibits tumorigenesis and metastasis of breast cancer cells.47 HRG is expressed in a significant proportion of human breast cancer biopsies and correlates with poor histological grade.48,49 Our earlier studies demonstrated that HRG-dependent activation of the Her-2, -3, and -4 network associates with increased breast cancer cell sensitivity to doxorubicin.10,50 However, it remained to be addressed whether HRG-induced transactivation of Her-2 can cause the same biologic responses than Her-2 overexpression with regards to sensitivity to chemotherapeutic agents. Herein we will address whether breast cancer patients susceptible to treatment with trastuzumab and/or chemotherapy should also include those with overexpression of HRG.
Autocrine HRG Induces Constitutive Phosphorylation of the erbB Receptors in Low Her-2 Expressing Breast Cancer Cells We examined HRG effects on Her-2 expression in MCF-7 cells, which express physiological levels of the Her-2. Densitometric analyses of immunoblotting of p185Her-2 revealed that Her-2 protein expression was up to 80% lower in cell lysates from two breast cancer metastatic models developed in our laboratory MCF-7 derived T7 and T8 clones,44,50 than in MCF-7/V control cells (Fig 1A). Equivalent results were obtained in the HRG retroviral vector model (MCF-7/pBABE and MCF-7/HRG cells). HRG overexpression resulted in a constitutive hyperactivation of Her- receptors. ErbBs-associated tyrosine phosphorylation was likewise undetectable in control cells (Fig 1A). Although the differences in the phosphorylation levels of the erbBs reflected a slight variation of HRG expression levels among the cells overexpressing HRG, it should be noted that erbBs-associated tyrosine phosphorylation in all HRG-overexpressing MCF-7 derivatives was comparable with breast cancer cells with HER-2 gene amplification and overexpressing (Fig 1B) as the SK-Br3 and BT-474 cells. While, exogenous HRG-induced downregulation of Her-2 more slowly than of the EGF receptor,51,52 our data clearly demonstrate that endogenous expression of HRG significantly decreases p185Her-2 levels in MCF-7 cells.
HRG Overexpression Induces Breast Cancer Cell Resistance to CDDP The metabolic status of CDDP-treated cells was initially evaluated using a MTT-based cell viability on T7 and T8 cells following a 5-day treatment concentrations of CDDP ranging from 5 µmol/L to 75 µmol/L. CDDP concentrations required to produce the median effect (dose required to produce the median-effect [Dm], analogous to the concentration of drug required for 50% inhibition of cell growth [IC50] value) were calculated using the Chou & Talalay approach.53 CDDP sensitivity was reduced up to five-fold in MCF-7/T7 and MCF-7/T8 cells and reduced up to six-fold in MCF-7/HRG cells relative to MCF-7/V and MCF-7/pBABE cells, respectively (Table 1). In addition, low concentrations of CDDP decreased colony formation of MCF-7 control cells (Dm = 5 µmol/L CDDP; Table 2). A significantly higher concentration of CDDP (> 50 µmol/L CDDP) was required to inhibit up to 14-fold the anchorage-independent growth of T7, T8, and MCF-7/HRG cells (relative to control cells; Table 2). These findings strongly suggest that HRG overexpression promotes resistance to CDDP-induced cytotoxicity.
Trastuzumab Cotreatment Synergistically Enhances CDDP Efficacy in HRG Overexpressing Breast Cancer Cells To investigate the influence of trastuzumab HRG-overexpressing MCF-7 cells to CDDP, we first determine T7, T8, and MCF-7/HRG cells response to trastuzumab. These cells reveal modest trastuzumab inhibitory activity (data not shown). To test whether any cooperative action occurs between trastuzumab and CDDP in these cells, trastuzumab was used at a dose which caused less than 20% reduction cell viability of the HRG-overexpressing cells. A synergistic cytotoxic effect was observed when the HRG-overexpressing cells were cotreated with trastuzumab and increasing concentrations of CDDP. Coexposure to trastuzumab and 10 µmol/L CDDP in T7 cells achieved a 66% reduction in cell viability, whereas trastuzumab and CDDP, as single agents, reduced cell viability by 18% and 23%, respectively (Fig 2A through C). T8 cells demonstrated a 48% reduction in cell viability, whereas trastuzumab and CDDP, as single agents, caused reductions of 10% and 13%, respectively (Fig 2A through C), MCF-7/HRG cells demonstrated a 53% reduction in cell viability, each agent caused reductions of 5% and 22%, respectively (Fig 2D and E). Undeniably, cotreatment with trastuzumab and CDDP induced a 6- to 19-fold increase in the efficacy of CDDP in HRG-overexpressing cells (Table 1).
It could be argued that synergistic interactions in cells treated with CDDP and trastuzumab cannot be accurately discriminated from additive or antagonistic interactions on the basis of these data alone. Although there is still controversy over which method is best for detecting true in vitro synergy between drug combinations, we performed isobologram transformations of multiple dose-response analyses.53,54 Representative isobologram presented graphically in Figure 3 clearly denotes a synergistic effect of trastuzumab plus CDDP in HRG-overexpressing MCF-7 cells. While these figures provide a graphical representation of the nature of interaction between trastuzumab and CDDP, the values of the mean combination index (CMI) for each cell line are also labeled. Student’s t tests were computed to evaluate whether significant differences in CMI mean values occurred as compared with a null hypothesized CMI of 1 (addition) and to formally evaluate whether antagonism or synergism was evident. At 30% (cytostatic) effect level, concurrent administration of trastuzumab and CDDP resulted in synergism, indicating that the amount of the two drugs necessary to reduce by 30% cell viability in each one of the T7, T8, and MCF-7/HRG cells was 0.633, 0.381, and 0.572 times as much as it would be required if they demonstrated additive actions. Trastuzumab coexposure induced a marked increase in the number of colonies formed by MCF-7 cells treated with CDDP. Conversely, trastuzumab and CDDP significantly enhanced the cytotoxicity of CDDP in T7 and T8 cells (up to four-fold) similarly in MCF-7/HRG cells (up to 17-fold; Table 2).
We hypothesized that reduced sensitivity to CDDP and the ability of trastuzumab to synergistically enhance CDDP efficacy in HRG-overexpressing cells were not simply the result of changes in cell proliferation, but actually a change in apoptotic cell death. Accordingly, cells were exposed to CDDP plus trastuzumab and cell death was determined by a quantitative assay that measures DNA fragmentation. The combination of trastuzumab and CDDP significantly enhanced apoptosis by more than 4-fold over CDDP alone in MCF-7/HRG cells. A two-way analysis of variance showed that concurrent exposure synergistically potentiated the apoptotic effects of trastuzumab and CDDP as single agents (P < .001). Trastuzumab coexposure decreased CDDP-induced apoptosis in MCF-7/pBABE cells and the results were statistically significant (P < .01; Fig 4). These findings reveal to the best of our knowledge, for the first time, that an equivalent synergistic interaction between trastuzumab and CDDP occurs when Her-2 is activated by HRG-induced transactivation as it has been demonstrated previously by receptor overexpression.20-23
Trastuzumab Treatment Differentially Regulates Her-2–Driven Transduction Cascades in HRG-Overexpressing Cells Trastuzumab promotes dimerization the p185Her-2, receptor internalization, and downregulation by accelerating endocytic degradation.55-58 To investigate how trastuzumab modulated CDDP sensitivity in MCF-7/HRG, we assessed p185Her-2/neu expression and phosphorylation. Trastuzumab induced downregulation of p185Her-2/neu and increased phosphorylation MCF-7/pBABE cells. In contrast, in MCF-7/HRG cells, trastuzumab induced a time-dependent inactivation of Her-2 with a maximum reduction of phosphorylated p185Her-2/neu at 72 hours (Fig 5A). Trastuzumab induced AKT constitutive hyperactivity in MCF-7/HRG cells, whereas AKT activity was undetectable in MCF-7/pBABE cells. Importantly, trastuzumab treatment rapidly reduced the activity levels of AKT by 95% or more when compared with those found in untreated MCF-7/HRG cells (Fig 5B). To test if this effect was linked to PI-3'K activity, we tested whether LY294002, a specific inhibitor of the p110 catalytic subunit of PI-3'K, to modulate AKT activation in a HRG-related manner. Similar to trastuzumab, LY294002-induced blockade of PI-3'K activity significantly reversed AKT hyperactivation in MCF-7/HRG cells. In addition, trastuzumab completely blocked hyperactivation of ERK1/2 in MCF-7/HRG cells, whereas a transient increase in ERK1/2 phosphorylation was seen in treated control cells (Fig 5B). These findings support the view that trastuzumab acts as an antagonist of Her-2 driven activation of antiapoptotic and proliferative cascades in cells exhibiting HRG-dependent activation of Her-2. Conversely, in the absence of autocrine HRG, trastuzumab acts as an agonist of the p185Her-2/neu-driven signaling in cells expressing physiological levels of HER-2/neu gene.
HRG-Promoted Trans-Phosphorylation of Her-2 Through Heterodimer Formation Our data demonstrating that trastuzumab-induced inhibition of signaling cascades in MCF-7/HRG cells challenges previous reports indicating that HRG-induced signaling was unaffected by trastuzumab but was sensitive to pertuzumab, an Her-2 antibody, that blocks heterodimerization of Her-2 with erbBs.59 Because that study was performed with MCF-7 cells treated with exogenous recombinant HRG, the interaction between HRG and erbB receptors may somehow be different from a transmembrane constitutively expressed HRG, as recently proposed.60 Because effective signaling from Her-2 requires interaction with other erbB receptors, especially Her-3,61 the sole modification in Her-2 activation may not be the only molecular mechanism underlying trastuzumab-induced REC in MCF-7/HRG cells.
We investigated whether secretion of HRG and subsequent activation of the erbB network are necessary molecular events for HRG-promoted chemoresistance and for trastuzumab-triggered REC effect in cells exhibiting HRG-induced activation of Her-2. HRG is a membrane-anchored growth factor consisting of a secreted extracellular domain, a transmembrane domain.62 The N-terminal domain of HRG is cleaved from the membrane and secreted into the intracellular space where it activates cells expressing the Her-2/3/4 receptors.62 To evaluate the relevance of membrane-anchored HRG, first, we generated structural deletion mutant of HRG that could not be secreted lacking the transmembrane domain of HRG and generated stable MCF-7/delta (
To further confirm this hypothesis we compared the efficacy of CDDP as single agent and in conjunction with trastuzumab against MDA-MB-231 and Hs578T cells, two highly metastatic breast cancer cell lines endogenously overexpressing HRG and expressing low levels of Her-2.43,47,63 Remarkably, trastuzumab coexposure significantly enhanced (two to three times) CDDP-induced cytotoxicity (Fig 7). We recently reported that blockage of HRG expression nearly completes reversion of the tumorigenic and metastatic phenotype of MDA-MB-231 cells in vivo.47 Our data clearly demonstrated that blocking HRG expression promotes a decrease in the activation status of downstream signaling molecules.47 Here, we observed that antisense blockade of HRG expression in MDA-MB-231 cells resulted in a significant increase in CDDP efficacy as the mean IC50 value decreased by three-fold in two representative antisense-HRG transfectants (AS-31 and AS-6 clones; Fig 7A). Similarly, two typical antisense-HRG transfectants derived from the Hs578T cell line (1F1 and 1D2 clones) were more sensitive to CDDP (up to 14 times) than were the control cells (Fig 7B). A synergistic sensitization was also observed when MDA-MB-231 and Hs578T cells were cotreated with trastuzumab and the taxanes paclitaxel, while paclitaxel efficacy was greatly enhanced in the antisense-HRG AS-31, AS-6, 1F1, and 1D2 clones (data not shown). These results arising from low Her-2 HRG-overexpressing cells serve as proof of the concept that: HRG acts as a potent promoter of breast cancer tumorigenicity and metastasis47; autocrine receptor activation by HRG actively contributes to chemosensitivity independently of Her-2 overexpression; and HRG-promoted Her-2 trans-phosphorylation through heterodimer formation provides a novel biomarker predicting newer responders following treatment with combination chemotherapy and trastuzumab.
Breast cancer cells engineered to overexpress HRG offer the distinct advantage of studying HRG-induced p185Her-2 receptor activation in the absence of Her-2 overexpression. Our current approach explores a unique mechanism linking HRG-induced transactivation of Her-2-driven cell signaling and chemotherapy efficacy. The conclusions from this study include that the DNA alkylator CDDP is more than 10 times less effective in low Her-2-expressing cells engineered to overexpress HRG. Suggesting that HRG-induced transactivation of p185Her-2 receptor is sufficient to determine greater breast cancer cell resistance to CDDP than Her-2 overexpression itself.13 In addition, trastuzumab synergistically enhances CDDP-induced cell killing in HRG-overexpressing breast cancer cells, a therapeutic advantage previously observed in Her-2-overexpressing breast cancer cells.21-25 The ability of trastuzumab to synergistically sensitize HRG-overexpressing breast cancer cells to chemotherapy is not restricted to CDDP but further occurs with paclitaxel and vincristine. Our data not only confirm that a considerable potentiation of chemotherapy efficacy occurs when combined with trastuzumab but further demonstrate that a REC effect, which has been suggested to specifically target cancer cells bearing Her-2 overexpression and has not effect on cells expressing physiological levels of the gene,21-25 is equally pronounced in HRG expressing and induces activation of Her-2 occurring in the absence of Her-2 overexpression. Trastuzumab did not induce significant cell growth inhibition in MCF-7 cells engineered to overexpress HRG. This is consistent with reports showing that HER-2 gene amplification is necessary for trastuzumab-induced cytotoxicity and apoptosis. Interestingly, we demonstrate that trastuzumab treatment of HRG-overexpressing breast cancer cells efficiently blocks HRG-dependent Her-2 phosphorylation and activation of the downstream antiapoptotic and proliferative transducers. This blockade, when combined with chemotherapeutic agents, results in a synergistic breast cancer cell killing. Although the precise biochemical events responsible for the connection between HRG-dependent breast cancer signaling and the trigger of a REC mechanism in the presence of trastuzumab have yet to be clarified, our current findings strongly suggest that trastuzumab efficiently impairs HRG-induced Her-2 to Her-3 heterodimers, thus depriving breast cancer cells of prosurvival pathways. Accordingly, trastuzumab cotreatment is ineffective in enhancing chemotherapy efficacy against MCF-7 cells engineered to overexpress a structural deletion mutant of HRG unable to function in an autocrine manner. Moreover, the combination of chemotherapy and trastuzumab produced a synergistic interaction in low Her-2 breast cancer cells endogenously overexpressing HRG. All together, these findings strongly support the concept that the impact of HER-2 oncogene on both the biologic behavior of breast cancer and the response to trastuzumab-based therapies may be better reflected when considering expression of HER-2 gene amplification and/or overexpression and also HRG expression, the active forms of erbB receptors, and the activation status of downstream transduction cascades. We demonstrated that among 189 samples with invasive breast carcinoma immunohistochemically examined for the presence of HRG, 57 samples (30%) were HRG overexpressors. When analyzed for Her-2 levels, only three of 57 HRG-positive tumors were found to be Her-2 overexpressors. Interestingly, when analyzed for the activation status of Her-2 using a phosphorylation state-dependent anti-Her-2 monoclonal antibody (PN2A), 67% of HRG overexpressors were found to exhibit low levels of Her-2 that, however, were in the phosphorylated status (Table 4). Because a substantially greater frequency of phosphorylated Her-2 (pHer-2; 58%) was observed in Her-2-overexpressing ductal carcinoma in situ, while pHer-2 occurs only in a minority of Her-2-overexpressing invasive breast carcinomas (12%), with only those cases with activated phosphorylated receptor displaying aggressive clinicopathologic features and adverse prognoses,37,38,64-67 our results support the concept that pHer-2 by Her-2 overexpression may play a critical role in early phases of breast tumorigenesis whereas it may be of lesser importance in the biology of invasive and metastatic breast cancer. Although we are lacking retrospective studies evaluating the response to trastuzumab treatment in breast cancer patients with known content of HRG and Her-2, and caution must be applied when extrapolating in vitro results into clinical practice, our findings strongly suggest that phospho-Her-2 receptor analyses, dual assays for HRG and pHer-2, or a triple test involving phosphorylated Her-2, HRG, and Her-2 will be more useful than a single assay to identify patients likely to respond to combined chemotherapy and trastuzumab. For example, while 32% of the tumor biopsies evaluated in our study were positive for Her-2, 52% of breast cancer patients positive for the combination analysis for HRG/phosphorylated Her-2 would benefit from trastuzumab-chemotherapy combination therapies.
In summary, two distinct breast cancer populations—one overexpressing Her-2 with low levels of HRG, and the other overexpressing HRG but not Her-2—appear to employ the same signaling pathways controlling breast cancer chemosensitivity. Although more studies are needed to definitely establish a role for HRG as prognostic and/or predictive marker in breast cancer, our current approach indicates that, in clinical practice, a correct molecular pathologic analysis of breast carcinomas should incorporate the detection of HRG and its autocrine activating actions on Her-2–driven signaling as clinical valuable markers in trastuzumab-based regimens. Indeed, our report sets the stage to molecularly define a much larger population of breast cancer patients for whom treatment with trastuzumab in combination with chemotherapy will be more efficient even in the absence of HER-2 gene amplification and/or overexpression.
The authors indicated no potential conflicts of interest.
We thank David Stern, PhD, and Michael DiGiovanna, MD, PhD, for providing the antiactivated Her-2 antibody and Adriana Papadimitropoulou for technical support.
published online ahead of print at www.jco.org on July 17, 2006. Supported by the Special Program for Research Excellence (P50CA89019-03) to R.L. Herceptin was provided by the Evanston Northwestern Healthcare Pharmacy (Evanston, IL). Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
) P < .001; NS, not statistically significant. All statistical tests were two sided.
) HRG cell line. Therefore, the 
