Trastuzumab in Combination With Heregulin-Activated Her-2 (erbB-2) Triggers a Receptor-Enhanced Chemosensitivity Effect in the Absence of Her-2 Overexpression

  1. Ruth Lupu
  1. 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
  1. 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
 
Next Section

Abstract

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.

Previous SectionNext Section

INTRODUCTION

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.

Previous SectionNext Section

METHODS AND RESULTS

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.

Fig 1.
View larger version:
Fig 1.

Autocrine heregulin promotes constitutive hyper-tyrosine phosphorylation of erbB receptors in MCF-7 cells. Testing for p185Her-2 expression and phosphor-tyrosine phosphorylation was performed as previously described.44

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.

View this table:
Table 1.

Anchorage-Dependent Sensitivity of HRG-Overexpressing MCF-7 Cells to CDDP-Induced Cytotoxicity

View this table:
Table 2.

Anchorage-Independent Sensitivity of HRG-Overexpressing MCF-7 Cells 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).

Fig 2.
View larger version:
Fig 2.

Trastuzumab cotreatment synergistically enhances cisplatin (CDDP) -induced cytotoxicity in heregulin -overexpressing breast cancer cells. CDDP was purchased from Sigma-Chemical (St Louis, MO). CDDP and trastuzumab were freshly prepared from stock solution and diluted with growth medium. CDDP sensitivity of cells growing in monolayer was determined using an MTT reduction assay.

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).

Fig 3.
View larger version:
Fig 3.

Analysis of the nature of the interaction between cisplatin (CDDP) and trastuzumab in MCF-7 cells engineered to overexpress heregulin (HRG). The combined effect of a simultaneous exposure to CDDP plus trastuzumab was evaluated by the isolobologram method (a dose-oriented geometric method of assessing drug interactions)54 using the the concentration of drug required for 30% inhibition of cell growth (IC30) values for MCF-7/T7, MCF-7/T8, and MCF-7/HRG cells. See Online Only Appendix.

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

Fig 4.
View larger version:
Fig 4.

Trastuzumab cotreatment synergistically enhances cisplatin (CDDP) -induced apoptosis in heregulin-overexpressing breast cancer cells. Quantification of apoptosis-related cell death was determined by Cell Death ELISA (Roche Biochemicals, Indianapolis, IN). The enrichment of histone-DNA fragmentation was expressed as fold increase in absorbance: [A405 –A490]TREATED/[A405-A490]UNTREATED. Data are the mean (columns) and 95% Cls (bars) of three independent experiments. One-factor analysis of variance was used to analyze differences in the percentage of apoptosis between the various treatment groups and the control group. (*) P < .01; (†) P < .001; NS, not statistically significant. All statistical tests were two sided.

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.

Fig 5.
View larger version:
Fig 5.

Differential signaling by trastuzumab in heregulin-overexpressing cells. (A) MCF-7/pBABE (top) and MCF-7/HRG (bottom) cells were treated with 20 μg/mL trastuzumab for 0, 24 hours, 48 hours, and 72 hours and tested in a p185Her-2 and P-tyrosine immunoblotting. (B) Overnight serum-starved MCF-7/pBABE and MCF-7/HRG cells were treated with 20 μmol/L LY294002 (2 hours), 20 μmol/L U0126 (2 hours), or 20 μg/mL trastuzumab (2 hours and 24 hours).

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 (Δ) HRG cell line. Therefore, the ΔHRG protein is sequestered into a cellular compartment and is not secreted, thus preventing the autocrine actions of HRG via erbBs.50 To simultaneously identify the activation status of each receptor of the erbBs network, we utilized a phospho-receptor tyr-kinase, sensitive and semi-quantitative protein array technology allows the profiling of receptor tyr-kinases. Third, we investigated how autocrine HRG altered breast cancer cell sensitivity to cytotoxic agents others than CDDP, including paclitaxel, vincristine, and fluorouracil. Phospho-receptor tyrosine-kinase profiling of MCF-7/HRG cells clearly revealed the ability of autocrine HRG to significantly activate Her-3, the Her-2, and to weakly activate Her-1. Trastuzumab reduces activation of Her-3, while completely inhibits phosphorylation of Her-2. In contrast, the ΔHRG failed to activate the erbBs receptor network in MCF-7 cells (Fig 6). When comparing the efficacy of CDDP, fluorouracil, paclitaxel, and vincristine as single agents or in conjunction with trastuzumab against MCF-7/HRG cells (Table 3), concluding that: MCF-7/HRG reveal crossresistance to fluorouracil (up to three-fold), paclitaxel (up to 10-fold), and vincristine (up to seven-fold); MCF/ΔHRG and MCF-7/pBABE cells do not have any resistance; trastuzumab synergistically interacts with CDDP, paclitaxel, and vincristine in MCF-7/HRG cells, whereas it fails to modulate chemotherapy efficacy in MCF/ΔHRG cells. These findings strongly suggest that, in breast cancer cells exhibiting ligand-induced transactivation of Her-2, trastuzumab efficiently interferes with the lateral signaling between Her-2 to Her-3 altering phosphorylation and preventing Her-2/Her-3 dimerization to function as an oncogenic unit.61

Fig 6.
View larger version:
Fig 6.

Trastuzumab treatment blocks autocrine actions of heregulin (HRG) on Her-receptors network. Overnight-serum starved MCF-7/pBABE, MCF-7/HRG, and MCF-7/delta HRG cells (untreated or treated with 20 μg/mL trastuzumab for 24 hours) were solubilized in NP-40 lysis buffer; 500 μg of lysates were diluted and incubated with the human phospho-receptor tyrosine kinase array kit (Proteome Profiler; R&D Systems, Minneapolis, MN) as per manufacturer's instructions.

View this table:
Table 3.

Impairment of HRG Autocrine Actions on the Activity of erbB Receptors Inhibits Trastuzumab-Induced REC in MCF-7 Cells

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.

Fig 7.
View larger version:
Fig 7.

Trastuzumab treatment enhances cisplatin (CDDP) efficacy in low Her-2 breast cancer cells endogenously overexpressing heregulin (HRG). Data presented are from HRG antisense clones (AS-31 and AS-6 for MDA-MB-231 cells and 1F1 and 1D2 for Hs578T). CDDP (the concentration of drug required for 50% inhibition of cell growth [IC50]) values for the antisense-HRG derivatives AS-31, AS-6, 1F1, and 1D2 were calculated as described in Figure 2 and Table 1. See Online Only Appendix.

Previous SectionNext Section

DISCUSSION

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.

View this table:
Table 4.

Relationship Between Her-2, Phosphorylated Her-2, and HRG

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.

Previous SectionNext Section

Authors' Disclosures of Potential Conflicts of Interest

The authors indicated no potential conflicts of interest.

Previous SectionNext Section

Author Contributions

Conception and design: Javier A. Menendez, Inderjit Mehmi, Ruth Lupu

Financial support: Ruth Lupu

Provision of study materials or patients: Ruth Lupu

Collection and assembly of data: Javier A. Menendez, Inderjit Mehmi, Ruth Lupu

Data analysis and interpretation: Javier A. Menendez, Ruth Lupu

Manuscript writing: Javier A. Menendez, Ruth Lupu

Final approval of manuscript: Javier A. Menendez, Inderjit Mehmi, Ruth Lupu

Previous SectionNext Section

GLOSSARY

Autocrine:
Secretion of a substance, for example, a growth factor, which stimulates the secretory cell itself.
Chou & Talalay approach:
A mathematical method used to analyze and define synergy. This method yields a parameter (the combination index) that describes the nature of the interaction between agents (antagonism, addition, and synergism) in a given combination. This method takes into account the potency (median-effect dose values or agent concentrations at 50% effect [IC50]) and the shape (sigmoidicity) of the dose-effect curve based on the median-effect equation.
Densitometric:
Densinometric analysis is measuring the optical density of the bands appearing in blotting techniques(Northern, Southern, Western, etc.) in the order to semiquantify gene, mRNA or protein expression.
Isobologram:
An isobologram is a means of graphically displaying drug interactions.
REC (receptor-enhanced chemosensitivity):
Synergistic activity of the anti-Her-2/neu monoclonal antibody trastuzumab when administered in conjunction with cytototoxic drugs.
Retroviral vector:
Artificial DNA construct derived from a retrovirus, used to insert genetic material into cells.

Previous SectionNext Section

Acknowledgments

We thank David Stern, PhD, and Michael DiGiovanna, MD, PhD, for providing the antiactivated Her-2 antibody and Adriana Papadimitropoulou for technical support.

Previous SectionNext Section

Footnotes

  • 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.

  • Received September 22, 2005.
  • Accepted April 5, 2006.
Previous Section
 

REFERENCES

  1. ↵
    Slamon DJ, Clark GM, Wong SJ, et al: Human breast cancer: Correlation of relapse and survival with amplification of the Her-2 oncogene. Science 235:177-182, 1987
    Abstract/FREE Full Text
  2. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the Her-2 proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989
    Abstract/FREE Full Text
  3. Ross JS, Fletcher JA: The Her-2 oncogene in breast cancer: Prognostic factor, predictive factor, and target for therapy. Stem Cells 16:413-428, 1998
    Medline
  4. ↵
    Yarden Y, Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127-137, 2001
    CrossRefMedline
  5. ↵
    Yu D, Liu B, Tan M, et al: Overexpression of c-erbB-2/neu in breast cancer cells confers increased resistance to taxol via mdr-1-independent mechanisms. Oncogene 13:1359-1365, 1996
    Medline
  6. Pegram MD, Finn RS, Arzoo K, et al: The effect of Her-2 overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 15:537-547, 1997
    CrossRefMedline
  7. ↵
    Yu D, Hung M-C: Role of erbB2 in breast cancer chemosensitivity. BioEssays 22:673-680, 2000
    CrossRefMedline
  8. ↵
    Paik S, Bryant J, Park C, et al: ErbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 90:1361-1370, 1998
    Abstract/FREE Full Text
  9. ↵
    Paik S, Bryant J, Tan-Chiu E, et al: HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National Surgical Adjuvant Breast and Bowel Project protocol B-15. J Natl Cancer Inst 92:1991-1998, 2000
    Abstract/FREE Full Text
  10. ↵
    Harris LN, Yang L, Tang C, et al: Induction of sensitivity to doxorubicin and etoposide by transfection of MCF-7 breast cancer cells with heregulin beta-2. Clin Cancer Res 4:1005-1012, 1998
    Abstract
  11. Harris LN, Yang L, Liotcheva V, et al: Induction of topoisomerase II activity after ErbB2 activation is associated with a differential response to breast cancer chemotherapy. Clin Cancer Res 7:1497-1504, 2001
    Abstract/FREE Full Text
  12. Yen L, Nie ZR, You XL, et al: Regulation of cellular response to cisplatin-induced DNA damage and DNA repair in cells overexpressing p185(erbB-2) is dependent on the ras signaling pathway. Oncogene 14:1827-1835, 1997
    CrossRefMedline
  13. ↵
    Alaoui-Jamali MA, Paterson J, Al Moustafa AE, et al: The role of ErbB-2 tyrosine kinase receptor in cellular intrinsic chemoresistance: Mechanisms and implications. Biochem Cell Biol 75:315-325, 1997
    CrossRefMedline
  14. ↵
    Carter P, Presta L, Gorman CM, et al: Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89:4285-4289, 1992
    Abstract/FREE Full Text
  15. Baselga J, Tripathy D, Mendelsohn J, et al: Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J Clin Oncol 14:737-744, 1996
    Abstract/FREE Full Text
  16. Cobleigh MA, Vogel CL, Tripathy D, et al: Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 17:2639-2648, 1999
    Abstract/FREE Full Text
  17. Mendelsohn J, Baselga J: Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 21:2787-2799, 2003
    Abstract/FREE Full Text
  18. ↵
    Vogel CL, Cobleigh MA, Tripathy D, et al: Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20:719-726, 2002
    Abstract/FREE Full Text
  19. ↵
    Slamon DJ, Leyland-Jones B, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpress HER2. N Engl J Med 344:783-792, 2001
    CrossRefMedline
  20. ↵
    Arteaga CL, Winnier AR, Poirier MC, et al: P185c-erbB-2 signal enhances cisplatin-induced cytotoxicity in human breast carcinoma cells: Association between an oncogenic receptor tyrosine kinase and drug-induced DNA repair. Cancer Res 54:3758-3765, 1994
    Abstract/FREE Full Text
  21. ↵
    Pietras RJ, Fendly BM, Chazin V, et al: Antibody to Her-2 receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells. Oncogene 9:1829-1838, 1994
    Medline
  22. ↵
    Pegram MD, Lopez A, Konecny G, et al: Trastuzumab and chemotherapeutics: Drug interactions and synergies. Semin Oncol 27:21-25, 2000 (suppl 11); discussion 92-100, 2000
  23. ↵
    Pietras RJ, Pegram MD, Finn RS, et al: Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene 17:2235-2249, 1998
    CrossRefMedline
  24. ↵
    Pegram MD, Lipton A, Hayes DF, et al: Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 16:2659-2671, 1998
    Abstract
  25. ↵
    Pegram MD, Slamon DJ: Combination therapy with trastuzumab (herceptin) and cisplatin for chemoresistant metastatic breast cancer: Evidence for receptor-enhanced chemosensitivity. Semin Oncol 26:89-95, 1999 (suppl 12)
    Medline
  26. ↵
    Baselga J, Norton L, Albanell J, et al: Recombinant humanized anti-HER2 antibody (herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 58:2825-2831, 1998
    Abstract/FREE Full Text
  27. Lee S, Yang W, Lan KH, et al: Enhanced sensitization to taxol-induced apoptosis by herceptin pretreatment in ErbB2-overexpressing breast cancer cells. Cancer Res 62:5703-5710, 2002
    Abstract/FREE Full Text
  28. Yu D: Mechanisms of ErbB2-mediated paclitaxel resistance and trastuzumab-mediated paclitaxel sensitization in ErbB2-overexpressing breast cancers. Semin Oncol 28:12-17, 2001 (suppl 16)
    Medline
  29. Esteva FJ, Valero V, Booser D, et al: Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer. J Clin Oncol 20:1800-1808, 2002
    Abstract/FREE Full Text
  30. Burstein HJ, Kuter I, Campos SM, et al: Clinical activity of trastuzumab and vinorelbine in women with HER2-overexpressing metastatic breast cancer. J Clin Oncol 19:2722-2730, 2001
    Abstract/FREE Full Text
  31. ↵
    Pegram M, Hsu S, Lewis G, et al: Inhibitory effects of combinations of Her-2 antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 18:2241-2251, 1999
    CrossRefMedline
  32. Pegram MD, Konecny GE, O'Callaghan C, et al: Rational combinations of trastuzumab with chemotherapeutic drugs used in the treatment of breast cancer. J Natl Cancer Inst 96:739-749, 2004
    Abstract/FREE Full Text
  33. ↵
    Pegram MD, Pienkowski T, Northfelt DW, et al: Results of two open-label, multicenter phase II studies of docetaxel, platinum salts, and trastuzumab in HER2-positive advanced breast cancer. J Natl Cancer Inst 96:759-769, 2004
    Abstract/FREE Full Text
  34. ↵
    Mendelshon J, Baselga J: The EGF receptor family as targets for cancer therapy. Oncogene 19:6550-6565, 2000
    CrossRefMedline
  35. ↵
    Lane HA, Beuvink I, Motoyama AB, et al: ErbB2 potentiates breast tumor proliferation through modulation of p27 (kip1)-Cdk2 complex formation: Receptor overexpression does not determine growth dependency. Mol Cell Biol 20:3210-3223, 2000
    Abstract/FREE Full Text
  36. ↵
    Xu FJ, Boyer CM, Bae DS, et al: The tyrosine kinase activity of the C-erbB-2 gene product (p185) is required for growth inhibition by anti-p185 antibodies but not for the cytotoxicity of an anti-p185-ricin-A chain immunotoxin. Int J Cancer 59:242-247, 1994
    Medline
  37. ↵
    DiGiovanna MP, Stern DF: Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor. Cancer Res 55:1946-1955, 1995
    Abstract/FREE Full Text
  38. ↵
    Thor AD, Liu S, Edgerton S, et al: Activation (tyrosine phosphorylation) of ErbB-2 (Her-2): A study of incidence and correlation with outcome in breast cancer. J Clin Oncol 18:3230-3239, 2000
    Abstract/FREE Full Text
  39. ↵
    Hudelist G, Kostler WJ, Attems J, et al: Her-2-triggered intracellular tyrosine kinase activation: In vivo relevance of ligand-independent activation mechanisms and impact upon the efficacy of trastuzumab-based treatment. Br J Cancer 89:983-991, 2003
    CrossRefMedline
  40. ↵
    Holmes WE, Sliwkowski MX, Akita RW, et al: Identification of heregulin, a specific activator of p185erbB2. Science 256:1205-1210, 1992
    Abstract/FREE Full Text
  41. ↵
    Alimandi M, Romano A, Curia MC, et al: Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 10:1813-1821, 1995
    Medline
  42. ↵
    Lewis GD, Lofgren JA, McMurtrey AE, et al: Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness. Cancer Res 56:1457-1465, 1996
    Abstract/FREE Full Text
  43. ↵
    Lupu R, Cardillo M, Cho C, et al: The significance of heregulin in breast cancer tumor progression and drug resistance. Breast Cancer Res Treat 38:57-66, 1996
    CrossRefMedline
  44. ↵
    Tang C, Perez C, Grunt T, et al: Involvement of heregulin-β2 in the acquisition of the hormone-independent phenotype of breast cancer cells. Cancer Res 56:3350-3358, 1996
    Abstract/FREE Full Text
  45. Krane IM, Leder P: NDF/heregulin induces persistence of terminal end buds and adenocarcinomas in the mammary glands of transgenic mice. Oncogene 12:1781-1788, 1996
    Medline
  46. ↵
    Atlas E, Cardillo M, Mehmi I, et al: Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo. Mol Cancer Res 1:165-175, 2003
    Abstract/FREE Full Text
  47. ↵
    Tsai MS, Shamon-Taylor LA, Mehmi I, et al: Blockade of heregulin expression inhibits tumorigenicity and metastasis of breast cancer. Oncogene 22:761-768, 2003
    CrossRefMedline
  48. ↵
    Dunn M, Sinha P, Campbell R, et al: Co-expression of neuregulins 1, 2, 3 and 4 in human breast cancer. J Pathol 203:672-680, 2004
    CrossRefMedline
  49. ↵
    Huang HE, Chin SF, Ginestier C, et al: A recurrent chromosome breakpoint in breast cancer at the NRG1/neuregulin 1/heregulin gene. Cancer Res 64:6840-6844, 2004
    Abstract/FREE Full Text
  50. ↵
    Atlas E, Bojanowski K, Mehmi I, et al: A deletion mutant of heregulin increases the sensitivity of breast cancer cells to chemotherapy without promoting tumorigenicity. Oncogene 22:3441-3451, 2003
    CrossRefMedline
  51. ↵
    Baulida J, Kraus MH, Alimandi H, et al: All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired. J Biol Chem 271:5251-5257, 1996
    Abstract/FREE Full Text
  52. ↵
    Nagy P, Jenei A, Damjanovich S, et al: Complexity of signal transduction mediated by ErbB-2: Clues to the potential of receptor-targeted cancer therapy. Pathol Oncol Res 5:255-271, 1999
    CrossRefMedline
  53. ↵
    Chou T-C, Talalay P: Quantitative analysis of dose-effect relationship: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27-55, 1984
    CrossRefMedline
  54. ↵
    Berenbaum MC: What is synergy? Pharmacol Rev 41:93-141, 1989
    Medline
  55. ↵
    Sliwkowski MX, Lofgren JA, Lewis GD, et al: Non-clinical studies addressing the mechanism of action of trastuzumab (herceptin). Semin Oncol 26:60-70, 1999
    Medline
  56. Ranson M, Sliwkowski MX: Perspectives on anti-HER monoclonal antibodies. Oncology 63:17-24, 2002 (suppl 1)
    CrossRefMedline
  57. De Santes K, Slamon D, Anderson SK, et al: Radiolabeled antibody targeting of the Her-2 oncoprotein. Cancer Res 52:1916-1923, 1992
    Abstract/FREE Full Text
  58. ↵
    Klapper LN, Waterman H, Sela H, et al: Tumor-inhibitory antibodies to HER-2/ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2. Cancer Res 60:3384-3388, 2000
    Abstract/FREE Full Text
  59. ↵
    Agus D, Akita R, Fox W, et al: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2:127-137, 2002
    CrossRefMedline
  60. ↵
    Yuste L, Montero JC, Esparis-Ogando A, et al: Activation of ErbB2 by overexpression of transmembrane neuregulin results in differential signaling and sensitivity to herceptin. Cancer Res 65:6801-6810, 2005
    Abstract/FREE Full Text
  61. ↵
    Holbro T, Beerli RR, Maurer F, et al: The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A 100:8933-8938, 2003
    Abstract/FREE Full Text
  62. ↵
    Weinstein EJ, Leder P: The extracellular region of heregulin is sufficient to promote mammary gland proliferation and tumorigenesis but not apoptosis. Cancer Res 60:3856-3861, 2000
    Abstract/FREE Full Text
  63. ↵
    deFazio A, Chiew YE, Sini RL, et al: Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines. Int J Cancer 87:487-498, 2000
    CrossRefMedline
  64. ↵
    DiGiovanna MP, Carter D, Flynn SD, et al: Functional assay for Her-2 demonstrates active signaling in a minority of Her-2-overexpressing invasive human breast tumors. Br J Cancer 74:802-806, 1996
    Medline
  65. DiGiovanna MP, Chu P, Davison TL, et al: Active signaling by Her-2 in a subpopulation of Her-2-overexpressing ductal carcinoma in situ: Clinicopathological correlates. Cancer Res 62:6667-6673, 2002
    Abstract/FREE Full Text
  66. ↵
    DiGiovanna MP, Stern DF, Edgerton SM, et al: Relationship of epidermal growth factor receptor to ErbB-2 signaling activity and prognosis in breast cancer patients. J Clin Oncol 23:1152-1160, 2005
    Abstract/FREE Full Text
  67. ↵
    Thor A: Are patterns of Her-2 amplification and expression among primary tumors and regional metastases indicative of those in distant metastases and predictive of herceptin response? J Natl Cancer Inst 93:1120-1121, 2001
    FREE Full Text
| Table of Contents

This Article

  1. Published online before print July 17, 2006, doi: 10.1200/JCO.2005.04.3489 JCO vol. 24 no. 23 3735-3746
  1. Abstract
  2. » Full Text
  3. PDF

Classifications

Related Content

  1. Related Editorial

Navigate This Article

Current Issue

  1. July 20, 2013, 31 (21)
  1. Alert me to new issues of JCO
    • Advertisement
    • Advertisement
    • Advertisement