The prognostic value of Her4 receptor isoform expression in triple-negative and Her2 positive breast cancer patients

The Her (human epidermal growth factor related) receptor tyrosine kinases (RTK) comprise four homologous proteins (Her1-4), which are differentially expressed during development and functional maintenance of the normal mammary gland [1‐4]. Spatiotemporally regulated RTK (co-)expression, however, is commonly disturbed in neoplastic mammary epithelium. 15% - 25% of breast cancers show Her2 receptor overexpression, which has a negative prognostic impact on the outcome of disease [5]. Specific Her2 receptor targeting with antibodies (e.g. trastuzumab and/or pertuzumab) or small molecule kinase inhibitors (e.g. lapatinib), usually applied in combination with chemotherapy or antihormonal therapeutic intervention, potentially prolongs the time to tumor progression and/or the overall survival rate of palliatively (metastatic) or (neo-)adjuvantly treated breast cancer patients [6]. Individual responsiveness, however, (based on Her2 overexpression/her2 gene amplification) cannot be predicted, varies significantly, and spans from de-novo to acquired resistance to moderate and high susceptibility [7].

Her1 and Her3 receptor expression in breast cancer has been described to be associated with a poor course and outcome of disease [8, 9]. In contrast, the prognostic (and predictive) value of Her4 receptor expression is uncertain [10‐16]. Both a positive and a negative impact of Her4 (co-)expression has been reported. This inconsistency can be conceivably attributed to the complex Her4 signaling capabilities, which among other reasons, might result from the differential expression of alternatively spliced Her4 isoforms [17, 18]. In fact, at least four different Her4 variants (JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, and JM-b/CYT2) can be generated by differential Her4 mRNA splicing. The juxtamembrane domain JM-a, but not JM-b, contains a cleavage site for the tumor-necrosis-factor-α-converting enzyme (TACE). CYT1/CYT2 intracellular domains have been demonstrated to differentially trigger intracellular signaling upon further Her4 activation by γ-secretase [19, 20]. Hence, the Her4 types differ in both function and signaling capabilities. Overall, not only four different Her receptors (Her1-4) but rather seven homologs (Her1-3 plus four Her4 isoforms) can potentially be coexpressed [17]. The prognostic value of isoform-related Her4 expression in breast cancer is, however, unknown.

The aim of this study was to evaluate the prognostic impact of Her4 isoform expression in well-characterized subgroups of breast cancer patients. Therefore, we analyzed the differential expression in primary tumor tissues of so-called triple-negative breast cancer (TNBC, i.e. estrogen, progesteron and Her2 receptor-negative) and Her2 positive patients by quantitative real-time polymerase chain reaction (qPCR). Isoform-specific Her4 expression was correlated with the outcome of disease in terms of event-free and overall survival. Extensive statistical analysis was applied to evaluate the prognostic value of Her4 (isoform) expression in well-defined TNBC and Her2 positive breast cancer cohorts.

We performed a Her4 isoform-specific expression analysis in 76 TNBC and 96 Her2 positive tissues of female tumor patients. If available, the associated non-malignant tissues were examined in addition (matched pair analysis, n = 26).

The impact of Her4 RTK expression on the course and outcome of breast cancer disease remains largely unclear. A number of findings emerged implying a favorable effect of Her4 expression [10‐13, 16]. In contrast, in-vitro and in-vivo studies demonstrated inhibited tumor cell proliferation by downregulation of Her4 expression or deactivation of Her4 function upon Her4 targeting [22‐24]. The retrospective study we present here reveals for the first time a favorable impact of Her4 expression on the OS of TNBC patients. In addition, we confirmed previously described indications for a beneficial impact of Her4 in Her2/ER positive patients [16]. A differential expression of Her4 isoforms does not, however, play a critical role in the course and outcome of these breast cancer subgroups.

In a multivariable Cox model with known strong predictors for OS and EFS such as age, grading and staging, Her4 expression was, however, no longer significant. This is not surprising since we were limited by the number of events in both collectives and the power to detect a significant effect of Her4 expression against other strong predictors is too low. Nevertheless we think that Her4 expression might still have a significant, independent effect on EFS and OS, which can only be demonstrated by an analysis of a larger cohort.

Accumulating data derived from preclinical investigations suggest that the apparent inconsistency regarding the importance of Her4 expression could be potentially explained by an ambivalent Her4 function i.e. pro-apoptotic [25, 26] and pro-proliferative [26, 27] activity. A tumor suppressive or oncogenic Her4 receptor activity might be attributed to receptor isoforms respectively expressed. Only the JM-a but not the JM-b extracellular domain is known to be ligand-independently activated by TACE-induced cleavage [18, 22, 27, 28]. Subsequently, the intracellular domain (either CYT1- or CYT2-4ICD) can be cleaved by γ-secretase and differentially triggers downstream signaling pathways. Once released, the 4ICD differentially triggers downstream signaling pathways e.g. by translocation into the nucleus and coactivation of ER-related gene transcription, which in turn stimulates cell proliferation [2, 29]. Alternatively, the Wwox protein would rather inhibit 4ICD routing into the nucleus. If not degraded by the ubiquitin ligase Itch, soluble 4ICD has been shown to interact via its BH3 subdomain with pro-apoptotic proteins (e.g. BAK) followed by increased permeability of mitochondria, cytochrom-c release, and finally cell death [15, 20, 25, 27].

Although Her4 inherently possesses a potential bivalent activity, the expression analysis of this study suggests a favored evolvement of a tumorsuppressive activity rather than oncogenic action. This observation is supported by the finding of reduced Her4 expression in rather progressive and poorly differentiated breast tumors as revealed by our data (Figure 1B) and other studies [4, 27]. Moreover, a reactivation of epigenetically silenced Her4 has been reported to induce apoptosis in breast cancer cells [30].

In Her2 positive breast cancer tissues we identified Her4 to be preferentially expressed in ER-positive rather than in ER negative specimens (Figure 3). This observation is in agreement with findings previously reported by Junttila et al. [22] and recently confirmed by Fujiwara et al. [31]. Obviously, the Her4 receptor develops its favorable impact primarily in the presence of ER, which in turn suggests a functional Her4 (4ICD)/ER interaction. This consideration is supported by the observation that the favorable impact of Her4 expression loses its significance in the Her2 positive/ER negative collective, both in terms of EFS (p = 0.370) and OS (p = 0.120). In contrast, the outcome (OS) of TNBC patients, who are typically ER negative, is significantly better when the tumor specimens appear Her4 positive (p = 0.030). Taking these findings together, the evolvement of a favorable (tumor suppressive) impact of Her4 expression in Her2/ER double-positive tumor patients is apparently inconsistent with a pro-proliferative activity that has been described in-vitro. Moreover, the Her4 receptor seems to restrain tumor growth even in the absence of ER expression, as shown for the TNBC collective. Within the period of observation, only 2 out of 12 Her4 positive TNBC patients suffered from a local recurrence. Accordingly, the favorable impact of Her4 expression is more pronounced in terms of OS (p = 0.03) than in terms of EFS (p = 0.257).

With respect to differential Her4 isoform expression, a preferred expression of CYT1 over CYT2 (or vice versa) intracellular domain, or a pronounced effect of high or low CYT1/CYT2 expression ratios cannot be concluded either from our data or other studies [22]. One might speculate that the functional diversity that has been attributed to the intracellular domain by pre-clinical studies [3, 32, 33], can either not be deduced by a descriptive study or does not, in fact, play a relevant role in-vivo. Instead, the identification of Her4 either by immunohistochemistry [10‐13], fluorescence in-situ hybridization (FISH) [14, 16], or qPCR [22] seems to be sufficient for attributing a positive impact on the course/outcome of breast cancer disease. Since JM-b isoforms are never expressed and CYT1/CYT2 intracellular domains are always simultaneously expressed, a diagnostic differentiation of Her4 isoforms is obviously not informative.

Considering a more translational approach, it could be evaluated to what extent the Her4 receptor represents a potential target that could be therapeutically utilized in 18% of TNBC and in 43% of Her2 positive breast cancers. As with ER, which basically represents a favorable prognostic marker as well, this hormone receptor is being very successfully targeted with e.g. tamoxifen or equivalent chemicals. Preclinical studies have revealed that anti-Her4 targeting with a newly developed antibody Ab1479 attenuates receptor activity and in turn reduces the formation of proliferative cell colonies [18, 24, 34]. Hence, even if the presence of a given biomarker (ER, Her4) is strongly correlated with a favorable outcome of disease, targeting this biomarker might be a potential beneficial therapeutic strategy.

Overall, our study reveals a positive impact of Her4 (JM-a) expression in triple-negative (OS) and Her2/ER-positive (EFS) breast cancer. The ever-growing body of evidence supporting the favorable impact of Her4 expression in breast cancer suggests the need to reexamine the commonly accepted idea that (over-)expression of (receptor) tyrosine kinases is necessarily associated with oncogenic activity. Only further extensive functional in-vitro and in-vivo analyses focusing on the importance of Her4 in the context of differential Her receptor co-expression will facilitate the consideration of this important receptor in individually optimized therapy based on a modular approach [35].