Breast cancer cell lines have been widely used as model systems for studies on breast cancer pathobiology and new therapy development [
]. Neve et al. reported that the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors [
]. The present study successfully employs HER2+ and HER2- breast cancer cell lines in optimization, characterization, and analytical specificity and sensitivity verification studies during the course of development of a novel functional signaling test. This work includes the IHC HER2+ clinical reference 3+ cell line SKBr3. We demonstrate that breast cancer cell lines and primary cells share many similarities regarding the phenotypic alterations (cell adhesion and temporal patterns) in response to HER family pathway agonists and antagonists when measured by CELx.
Defining and measuring receptor function using the CELx HSF test
Reliability, analytical specificity, sensitivity, and accuracy are essential prerequisites for the CELx HSF test to be considered for clinical diagnostic applications. When performing label-free biosensor-based viable cell assays, complexity is inherent and caution was exercised to test whether the signal was limited to a biological response resulting from a single molecule type binding to a single receptor type effecting signaling on a single pathway. In this study, a series of experiments were performed to demonstrate the selectivity and specificity of the assay for cell lines and primary cells.
FDA-approved HER2 inhibitors that treat HER2-positive breast cancer in clinical settings were used in this study to serve three purposes. First, the inhibitors helped to identify the specificity of the impedance signal arising from treatment of the cells with growth factors. Second, the anti-HER2 mAb inhibitors isolated the impedance signal arising solely due to HER2 participation in the growth factor activation of HER family pathway signaling. This provides a level of detail regarding the specificity of the selected reagents by using antagonists that work most proximal to signal initiation, receptor dimerization and receptor tyrosine kinase priming, thereby most effectively defining HER2 participation and isolating early signaling events before signal branching takes place. Finally, previous studies suggest differential sensitivities to the HER2 inhibitors among the cells lines used here [
]. Thus, the utilization of these HER2 signaling inhibitors would help to define the potential correlation of CELx signal with drug sensitivity in these cell lines.
The data for testing baseline effect of pertuzumab or lapatinib alone on cells (Fig.
, Panel a) indicate that neither have significant effect on SKBr3 cells in an HER2 overexpressing cell line. The same results were found when other HER2-overexpressing cell lines were tested and this result is in good agreement with published data indicating these drugs are cytostatic, not cytotoxic, and only slow cell passage through G
Trastuzumab was not selected for evaluation in this study because its primary mechanism of action, as reported by its manufacturer, is not HER2-driven signaling inhibition, but instead antibody-dependent cell-mediated cytotoxicity (ADCC). Any results studying the effect of trastuzumab on HER2-driven signaling would thus be confounded by the lack of direct linkage between the activity we are measuring, HER2 signaling, and trastuzumab’s primary mechanism of action (ADCC). Since the CELx HSF Test is designed to assess HER2 participation in HER family signaling, pertuzumab, a known HER2 dimer blocker, was selected instead to confirm the amount of HER2 participation in HER family signaling in this assay.
All HER2 CELx signals tested are agonist- and antagonist-concentration dependent within physiological doses in the picomolar to nanomolar range. When a HER2 antagonist (e.g. pertuzumab or lapatinib) is added with agonist, the cells show a significantly attenuated delta CI compared to the signal for addition of agonist only, indicative of a blocked HER2 signaling response. The work employs carefully selected components that have known specificity and well characterized affinity at concentrations that reduce the likelihood of activation of other pathways from high concentrations of agonists. EGF and NRG1b are very specific ligands for HER1 and HER3 receptors. Multiple literature references cite
receptor affinity of ~100pM for EGF and NRG1b [
]. This is in close agreement with the CELx test data presented here and in line with the concentrations that have been selected to measure agonism and antagonism in the CELx test.
Further dissection of the information from rich CELx data suggests sources of NRG1-driven test signal that is linked to more than just HER2/HER3 heterodimerization. In the SKBr3, HER2+ cells (Fig.
), lapatinib was able to reduce the NRG1 and EGF stimulation signals nearly to zero while pertuzumab was only able to attain partial (<50%) attenuation of the NRG1 and EGF-induced signals. The pertuzumab result indicates that HER2 was only partly involved as a heterodimer with HER1 and HER3 in the NRG1 and EGF stimulations and the remaining NRG1 and EGF signal could be indicated primarily for homodimer activity at HER1 and HER3, respectively. The lapatinib result on EGF stimulation of HER1 seems to confirm this. However, the lapatinib result on NRG1 signal cannot be explained quite as simply because HER3 is reported to possess only weak kinase activity and thus may be unable to generate very large signals [
]. This opens the possibility that HER3 binds NRG1 and heterodimerizes with HER1 or other receptor tyrosine kinases [
] to activate and sustain PI3K signaling or that HER3 expression is upregulated and its dephosphorylation is stalled; both are mechanisms that have been described previously [
]. This result highlights the difficulty of making limited protein time point analyses to determine drug efficacy and points to the value of a functional activity test such as the CELx HSF.
Determining pathway involvement
Next, a determination was made that the HER2-associated downstream signaling pathways controlling the cellular responses were quantified by the CELx HSF test. A series of pathway deconvolution experiments were performed using specific agonists and antagonists of different pathway members. The MAPK and the PI3K/AKT pathways are the two major pathways downstream of all HER family receptors [
]. Ligand binding, receptor phosphorylation, and receptor-intrinsic kinase activation in normal cells leads to the propagation of signals that regulate important cellular processes such as cell adhesion, migration, proliferation, and survival [
]. The present study focuses on PI3K and MAPK pathways and dissects the signaling mechanistically related to the HER2-driven phenotypic alterations. In both breast cancer cell lines and primary cancer cells, the data show that PI3K, not MAPK, is the downstream effector that contributes most significantly to the ligand-driven HER2 signal in the CELx HSF test for these cancer cell samples. This finding suggests that HER2 heterodimers, especially HER2/HER3, that form as determined by the use of a HER2 dimer blocker, are probably dominant in these types of breast tumors. The findings from the current study are in agreement with the existing literature, which suggests a high level of PI3K signaling in a subset of breast tumors and that HER2/HER3 is a strong driver of oncogenic HER2 signaling through PI3K activation in this subset [
The PI3K pathway is a highly complex signal progression model even though the pathway is often described in terms that imply otherwise. Multiple positive and negative effector proteins and mechanisms of PI3K pathway function and dysfunction have been demonstrated to attenuate and direct inhibition of PI3K activity in different patients. For example, mutations of PI3K combined with copy number variants or RAS activation and heritable cell-to-cell variability can affect the efficacy of inhibitors [
]. Therefore, it is not unexpected that incomplete response to PI3K inhibition would be seen in different patients. GSK1059615 on breast cancer cell lines
inhibits the phosphorylation of Akt at S473, with an IC50 of 40 nM [
], which translates well to the cellular IC
potency we find for the compound’s attenuation of signaling.
The CELx HSF test detects unexpected signaling and drug sensitivity in a HER2- breast cancer cell line. BT-483 is defined as having a PI3K activating mutation, E542K [
]. This activating mutation has been reported to act as a resistance mechanism [
] to HER2 signal inhibitors in HER2 overexpressed cell lines, which is speculated to explain the mutation’s correlation with poor prognosis. Despite having only normal expression levels of HER2 receptor, BT-483 recorded very high levels of NRG1b initiated PI3K initiated activity that was almost completely inhibited by pertuzumab and lapatinib. In fact, BT-483’s HER2-driven signaling activity was higher than activity found in all of the HER2+ cell lines evaluated (Fig.
). This finding suggests a more complex role for PI3K mutation as a resistance mechanism for HER2 signal inhibition. Other HER2-negative cell lines tested in this study, such as MCF-7 and MDA-MB-361 also have similar (E545K) PI3K activating mutations. However, the HER2-driven signaling test measured in these cell lines was consistent with normal pathway activity. This suggests that the high NRG1b initiated PI3K activity in BT-483 cells is not related solely to this PI3k mutation.
Endpoint cell-based assays provide a one-time “snapshot” of a focused biological event (e.g., phosphorylation of HER2 at a single time point). Although protein or gene based assays provide incremental information, they are still classical endpoint assays that reflect only the relative activity of a limited set of proteins that may be involved in disease propagation, and the results do not describe the dynamics or real-time status of the complete HER2 signaling network in a particular patient. Given that infinite permutations of circumstances are present and each persons’ genomic or proteomic status does not yet describe the
nature of that individual’s disease, a truly functional dynamic analysis may be more appropriate. Furthermore, allosterism, differential transient phosphorylation, signaling crosstalk, and a myriad of mechanisms of drug effect may contribute to the quantitative and qualitative activity of the HER2 signaling pathway in any particular patient [
]. Mylona et al. report on opposing effects of multisite phosphorylation shaping a signaling protein response to activation [
]. They conclude that their “results challenge the common assumption that multisite modification events act unidirectionally and can only be reversed or limited by antagonistic enzymes such as phosphatases.” The Mylona et al. study brings into doubt what is already suspected about the utility of correlations built upon single time point, limited site protein phosphorylation analyses’ for assessment of pathway function in whole cells. Santarpia et al. review biomarker studies in breast cancer and conclude: “It is likely that it is the combined effect of all genomic variations that drives the clinical behavior of a given cancer [
]. Furthermore, entirely new classes of oncogenic events are being discovered in the noncoding areas of the genome and in noncoding RNA species driven by errors in RNA editing. In light of this complexity, it is not unexpected that, with the exception of HER2 amplification, no robust molecular predictors of benefit from targeted therapies have been identified.” These factors contribute to the difficulty in using a protein quantification readout to comprehensively quantify signaling pathway regulation that relates drug response and therapeutic outcome prediction [
To verify the CELx HSF test concept, HER2+ (
9) and HER2- (
10) breast cancer reference cell lines were chosen. Fluorescence flow cytometry measurement of HER2 protein expression levels demonstrated HER2 expression data largely consistent with published CCLE data on
gene copy number in these cell lines [
]. However, the HER2 signal function determined by CELx HSF tests did not show any correlation with HER2 expression levels in these cell lines. The CCLE database documents HER2+ cell lines that are not responsive to HER2-targeted drugs
. Recent retrospective analyses of previous clinical trials indicated that there is no significant correlation between
gene copy number or total HER2 protein and clinical benefit from trastuzumab [
], although the molecular basis remains unclear and could be very diverse amongst patients. The results obtained from the present study suggest that some HER2+ breast tumors may not respond to HER2-signal inhibitors because they do not actually exhibit increased HER2 signaling activity or functional dependence on HER2 signaling, whereas some HER2- breast tumors could benefit from HER2-signal inhibitors because the HER2 pathway is abnormally active in these tumors. Collectively, the present data strongly suggests that HER2 signaling pathway dysfunction is the critical prerequisite for determining whether tumor cells respond to HER2-signal inhibitors.
The present test seeks to identify HER2-negative samples that have abnormally overactive HER2 signaling. Previous work by others have presented results that describe elevated protein ligands [
] of the HER family as the most likely cause of the PI3K activation in HER2 negative patients. The CELx test results with exogenous ligand equally applied to all samples suggest that there are other more systemic causes besides abundance of ligand. Other published work proposes elevated HER3 expression in HER2-negative cancers as leading to abnormal signaling in HER2 negative patients [
]. Several authors propose increased expression of HER2 in cancer stem cells to explain HER2– patient abnormal signaling or responsiveness to HER2-targeted therapy [
]. The flow cytometry data presented here do not support any of these receptor overexpression mechanisms.
Taken together, the results in this study demonstrate that the CELx HSF test is a selective and specific assay for monitoring the dynamic cellular pathway signaling status in live cells in response to ligand–receptor interactions and between receptors and receptor-targeting drugs. Functional assessment of HER2 signaling in live tumor cells with the CELx HSF test represents a possible new approach to diagnosing HER2-driven cancer in individual patients who have normal HER2 expression levels. It is envisioned that this test would be deployed in a central lab, where patient tumor specimens would be delivered and tested. To be successful, greater than 80–90% of clinical specimens must yield test results. To further develop this method, analytical validation studies meeting CAP (College of American Pathologists) and CLIA (Clinical Laboratory Improvement Amendments) established guidelines for Laboratory Developed Tests would be required. Finally, the clinical validity of using HER2-driven signaling activity as a diagnostic biomarker must be confirmed in a clinical trial that evaluates whether HER2-breast cancer patients with abnormal HER2-driven signaling benefit from treatment with HER2 signal inhibitors.