Introduction
Breast cancer is a complex disease, and depending on the molecular profiles of the tumor, it can be classified into several distinct intrinsic subtypes with different prognostic outcome [
1,
2]. The overall tumor biology of breast cancer subtypes is highly dependent on the expression of estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). One of the most fundamental clinical distinctions between breast cancer subtypes is whether the tumor responds to growth signaling through the hormonal (ER/PR) or HER2 receptors, as these tumors can be targeted with modern anti-hormonal or anti-HER2 therapy, respectively. Positivity for ER, PR and HER2 has therefore become highly important in the clinical management of breast cancer, both in the neoadjuvant/adjuvant and metastatic setting of the disease. Approximately 70% breast cancers are hormone receptor (HR) positive, meaning that they either express ER and/or PR to some extent [
3]. In ER+ breast cancers, PR is often used as a positive prognostic marker of disease outcome [
4], but the role of PR signaling in these cancers, still remains unclear. In advanced breast cancer, around 65% of all HR-positive breast cancer patients respond well to endocrine therapy [
5,
6], whereas patients that are ER positive and PR negative (ER+/PR−) have significantly worse prognosis [
7‐
9]. The PR is an ER-regulated gene, and ER+ tumors are usually also PR+, whereas ER- tumors are usually PR−. Therefore, single HR-positive (i.e., ER+/PR− or ER−/PR+) tumors represent only a small subgroup of breast cancer cases [
10]. While the majority of tumors are HR positive, nearly 15–20% of all breast cancer patients exhibit HER2 overexpression or HER2/
neu gene amplification. HER2− positive (HER2+) tumors are very heterogeneous, and in many cases, the overexpression of HER2 is associated with the loss of both ER and PR expression, but for 10% of breast cancers, both ER and HER2 are co-expressed [
11]. Women with both HER2- and HR-positive cancers do have a tendency to exhibit early resistance towards endocrine therapy [
12]. However, the prognostic impact of HER2 in breast cancer and the effectiveness of its drug target are well established in breast cancer patients, whereas the role of other protein tyrosine kinases (PTKs) in the loop of networks in ER+ breast cancer patients is rather unknown. PTKs have for a long time been considered as potent targets for the treatment of cancer as they are important mediators of signaling cascades, and may facilitate tumor progression. Nearly half of the tyrosine kinase complement is deregulated in cancer [
13], which has led to the recognition of deregulated PTKs as potential biomarkers for stratifying patients to personalized treatments [
14,
15]. In this study, we report a comprehensive analysis of tyrosine kinase activity in HR-positive breast tumors that are either HER2+ or HER2−. To our knowledge, profiling of a large number of PTKs in primary breast cancer specimens has not previously been performed, and will add valuable information regarding the aspect of molecular profiles of breast tumors, and will be important in identifying important biomarkers for clinical intervention.
Discussion
Since the clinical success of HER2-targeted therapies, like trastuzumab and pertuzumab, there has been an increased interest in PTKs as potentially attractive intracellular targets for breast cancer treatment. While HER2 has a profound impact on breast cancer biology and prognosis, one might expect that differences in HER2 status alone would have a significant effect on the activity of downstream kinases in ER+ breast cancers. However, when analyzing the phosphorylation patterns of kinases in ER+ breast cancer samples and cell lines, we observed no significant differences in phosphorylation of kinase substrates that could be correlated to HER2 status. However, it is important to mention that activation of HER2 may occur through several mechanisms in breast cancer [
33]. For instance, it has been reported that there are rare types of lung cancers that have HER2 kinase domain mutations that confer increased kinase activity without overexpression [
33]. This could potentially explain why we did not observe any differences between HER2+ and HER2− in our dataset, at least for the analysis involving phosphorylation of kinase substrates.
The role of estrogens and ER in breast cancer is undisputed, and drugs inhibiting estrogen synthesis or ER itself are effective cancer therapies for HR-positive tumors. However, the action of PR in breast cancer is still somehow underexplored and remains controversial. PR expression is induced by the activation of ER, and PR-related signaling pathways have important roles in the induction, progression and maintenance of neoplastic phenotype in breast cancer [
34]. Previous research also shows that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behavior by controlling chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions [
24]. Although it seems like PR appears to have a profound effect on prognosis and aggressiveness of breast cancer, the kinases and pathways that come to play is less studied.
In this study, we identified PR as a plausible factor responsible for differences in kinase activity within ER+ tumors. Higher kinase activity was observed in ER+/HER2−/PR− tumors compared to tumors that are positive for PR expression. Furthermore, using gene expression data from the METABRIC cohort [
23], we revealed that the loss of PR expression had a pronounced effect on survival in ER+ patients in general, but the differences were even more significant in ER+/HER2− patients, even when including a 24 kinase-encoding gene signature only. Using the 24-kinase-encoding-gene signature, we identified two clusters with differences in survival in ER+/HER2− patients. Cluster 1, which consisted mostly of PR+ patients, exhibited better survival than patients in cluster 2, which were mostly PR-. Interestingly, the patients within cluster 2 that were PR+ had worse survival compared to patients with same clinical features in cluster 1, indicating that the expression of kinase-encoding genes themselves might indicate worse survival altogether. Our data are so far in agreement with previous studies, which have shown that clinically, the PR status exerts a significant impact on the prognosis of ER+ breast cancer patients in which patients with PR- tumors have a worse outcome than PR+ tumors [
35‐
37]. Prat et al. [
38] also reported that a cut-off value of > 20% PR tumor cell positivity is a significant prognostic factor within luminal-type breast cancers. Also, the extent of PR expression has shown to be a potent prognostic indicator that can aid evaluations of the long-term prognosis of ER+/HER2− breast cancers [
34]. However, in our study, probably due to the small sample set, we could not observe differences in phosphorylation in patients with high or low PR tumor cell positivity, and as for the gene expression data, such differences in PR levels were not available. One might speculate that the PR+ patients that clustered together with patients that were PR- and vice versa could be due to differences of the amount of PR− positive tumor cells.
Furthermore, we identified four kinases that were important for the differences between PR+ and PR- in ER+/HER2− tumors, namely FGFR4, LCK, FRK and MST1R. These four kinases were identified through upstream analysis as potential kinases that might be responsible for changes between PR+ and PR− in ER+/HER2− tumors, but they were also identified in the METABRIC gene expression dataset as significantly differentially expressed between these two groups. Interestingly, we could not find any significant genes (except PGR) or kinases that were different between PR+ and PR− in ER+/HER2+ tumors. One might postulate that in ER+/HER2+ tumors, the HER2 pathway may be the major driver for tumor growth and hence there are less obvious differences observed between PR+ and PR- tumors.
FGFR4 and LCK had higher expression in HER2−/PR− samples, whereas FRK and MST1R had lower expression in HER2−/PR− samples. This was validated in breast tumor tissue through RT-PCR, except for MST1R. We also identified these kinases to be regulated directly by PR itself through PR chip-sequencing data. By adding progesterone to MCF7 cells (PR+), we observed that both FGFR4 and LCK were reduced in expression, whereas FRK and MST1R were increased. This is in concordance with our study where we see the complete opposite happening in PR- tumors. These findings are in the agreement of the idea that PR controls the expression of these kinases in ER+/HER2− tumors, and the deregulation of their activity might confer bad prognosis, and be important in treatment.
While PR+ tumors are associated with better prognosis in ER+ breast tumors [
4], the loss of PR expression is associated with differential breast tumor responses to anti-ER therapies [
7‐
9]. However, the role of PR activation seems to be a bit controversial, because in some studies, PR activation seems to promote breast cancer, whereas in some studies, progesterone treatment has been shown to be anti-proliferative in ER+/PR+ breast cells [
39‐
41]. At the same time, previous studies have shown that PR itself can be phosphorylated and activated independent of progesterone, through different growth factors and pathways, in particular through MAPK and PI3K/AKT pathway [
42]. Experimental data have even implied that growth factor signaling mediates PR downregulation through the activation of the PI3K–Akt–mammalian target of rapamycin (mTOR) pathway [
7].
Because PR is regulated by estrogen through ER, the ER-mediated signaling is important in ER+/PR+ tumors. However, in ER+/PR− tumors, one might hypothesize that they rely on other signal transduction pathways to grow because ER-mediated signaling is less important in these tumors. Therefore, the findings that more kinases were identified as differently expressed between PR+ and PR- in ER+/HER2− cancer is perhaps not completely unexpected. However, ER+/PR- cell lines are still dependent of ER for tumor growth. If ER is depleted in these cell lines, then the tumor growth is inhibited. The same happens with hormone resistant cell lines (most of them are PR-). In the case of ER+/PR− patients, they are less sensitive to hormone therapy but still responsive [
37]. The findings of the work in this study reveal that the loss of PR function leads to an increase of activity (and in some cases increase of expression as well) of kinases (different to HER2). This effect is not influenced by estrogen-ER activation. Hence, we might believe that the action is dependent of PR. However, we also know that ER, PR, and HER2 are related to each other, and gene signaling occur through a loop of network, which make further studies a necessity to understand their importance in cancer.
The results obtained in this study clearly demonstrated that the Ras/MAPK and PI3K pathways are important, and highly active in ER+/HER2− tumors lacking PR. When analyzing gene expression data, the PI3K cascade does not come up as significant between ER+/HER2− tumors with differential PR status, although, tyrosine kinase activity and immune pathways seems to be much more prominent. The activation of the PI3K pathway and its downstream targets is rather complex. Especially breast cancer tumorigenesis is believed to depend on the PI3K pathway because the majority of breast cancer cases harbor at least one mutation that potentially affects this pathway [
34]. Furthermore, the PI3K pathway is commonly altered in ER+/HER2− and ER+/PR− cancers [
37,
43]. The PI3K pathway is activated through phosphorylation of several proteins, among them also the four tyrosine kinases that we have identified in this study.
MST1R encodes for the protein RON, which is a RTK for the ligand macrophage-stimulating protein (MSP, MST1). Aberrant expression of RON is associated with tumor progression in breast cancer through its involvement with PI3K, MAPK, JNK, β-catenin, and STAT pathways [
44]. Overexpression of RON is associated with cell dissociation, motility and matrix invasion, which all are surrogate markers of an aggressive cancer phenotype with metastatic potential [
45]. In our study, the MST1R gene was lower expressed in PR− vs PR+ in ER+/HER2− in the METABRIC dataset, but could not be validated. Since this gene is a marker of invasion, and all our samples were primary tumors, this could possibly explain the reason why we could not see difference in expression, and it could most likely be considered as a marker of late stage disease.
FRK interacts and phosphorylate the tumor suppressor protein PTEN, and through its activation inhibits PI3K/AKT signaling pathway [
29]. Low levels of FRK show increased cell growth, colony formation and tumor growth, whereas high levels suppress tumor growth in breast cancer cell lines [
46]. In our study, FRK is lower expressed in PR− tumors compared to PR+ in ER+/HER2− tumors, making it an interesting marker for prognosis and potentially drug interventions.
Phosphorylated FGFR4 recruits and phosphorylates two important intracellular targets, phospholipase γ (PLCγ) and FGFR substrate 2 (FRS2). The MAPK and PI3K/AKT pathway can then be triggered by activated FRS2 through recruitment of growth factor receptor bound 2 (GRB2). Previous research has shown that high levels of FGFR4 mRNA could be an independent predictive factor, as high levels of FGFR4 show shorter progression-free survival in breast cancer patients treated with tamoxifen [
30,
47]. The FGFR4 expression levels also seem to be significantly increased in doxorubicin-resistant breast cancer clones [
48].
LCK itself is mostly found in T cells, and is involved in recruitment and activation of proteins, such as PLC and PI3K, that in turns activates the MAPK and PI3K/AKT pathway [
31]. The fact that genes involved in immunity were prominent in PR- compared to PR+ in ER+/HER2− tumors, makes LCK an important target, as tumor infiltrating lymphocytes (TILs) are important components of the microenvironment of the cancer. In breast cancer, they are associated with clinical outcomes as high TIL score is associated with worse prognosis and they provide prognostic information and can predict the patient’s response to neoadjuvant chemotherapy [
32]. In our study, both FGFR4 and LCK were found to be higher expressed in HER2−/PR− tumors vs HER2−/PR+. In HER2+ tumors, LCK and FGFR4 seems to be downregulated rather than upregulated in PR- tumors, which is the opposite to what we see in HER2− tumors. The fact that this overexpression is only observed in ER+/HER2− samples and not in ER+/HER2+ samples, seems to agree with what we find, that HER2− tumors are indeed much different from HER2+ tumors, and that HER2−/PR− are more “aggressive” or “oncogenic” in that sense. It could also mean that HER2 together with PR regulate their expression. One could postulate that when HER2 is present, HER2 could be more dominant than PR, and thereby the oncogenic events might occur through different genes/pathways. However, when HER2 is missing, other genes and pathways are more prominent.
As several studies have pointed out the importance of the PI3K pathway during resistance to endocrine therapy in breast cancer patients (39), together with the fact that ER+/PR+ breast cancer responds better to endocrine therapy compared to ER+/PR- breast cancer [
35‐
37], makes it of uttermost importance to study the consequences of the loss of PR in ER-positive breast cancers. The kinases LCK, FRK, FGFR4 and MST1R, all interacting with proteins within the PI3K pathway, seem to be a good starting point according to our findings.
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