PLC-gamma-1 phosphorylation status is prognostic of metastatic risk in patients with early-stage Luminal-A and -B breast cancer subtypes

Breast cancer incidence accounts for approximately 30% of all new cancer cases and 14% of all cancer-related deaths among women worldwide [1]. Implementation of screening programmes can detect early stage, node-negative (N0) tumours at low-risk of relapse, while advances in adjuvant treatments can promote decreased mortality from breast cancer [2, 3]. Nevertheless, almost 80% of women diagnosed with early stage disease are currently treated by breast-conserving surgery alone or combined with adjuvant therapy [4]. In these patients without nodal metastasis (N0), identification of high-risk patients for breast cancer relapse through use of established prognostic factors (i.e., age, tumour size and grade, hormone receptor status) cannot predict prognosis accurately. Moreover, nearly 90% of patients with cancer limited to the breast receive adjuvant treatments, although only 30% of these patients will ultimately relapse [5‐7]. Currently, adjuvant chemotherapy regimens are standard of care for treatment of early-stage disease that is oestrogen receptor (ER)-negative or human epidermal growth factor receptor (HER)2-positive. However, selecting chemotherapy for patients with ER-positive, HER2-negative disease is a more challenging task, due to the different risk profiles for disease relapse associated with this tumour subtype. Indeed, among patients with luminal tumours, there will be women at low risk of recurrence who will derive little benefit from chemotherapy combined with hormone therapy, and women at high-risk of recurrence where chemotherapy would be helpful. As far as the economic impacts on health care systems, is should be noted that adjuvant treatments are not devoid of toxicity. On this basis, above the search for new discriminatory biomarkers capable of selecting patients at risk of early relapse remains mandatory.

Among the lipid signalling metabolites in cells, the phosphoinositides are the most widely studied lipids due to their involvement in several cell signalling pathways. In particular, phosphoinositide-specific phospholipase C gamma 1 (PLCγ1) signalling is necessary for many physiological cellular processes (e.g., cell proliferation and differentiation) [8]. PLCγ1 is highly expressed in various tumours, including breast cancers [9‐11]. We previously observed that PLCγ1 down-regulation strongly reduced formation of MDA-MB-231-derived lung metastases in nude mice [12]. Furthemore, lung metastasis formation from prostate cancer cells was significantly reduced by a dominant-negative fragment of PLCγ1 [13].

In human, using tumour cases as training and validation sets, we have shown that overexpression of activated PLCγ1 is a risk factor for distant relapse in T1-T2, N0 breast cancer patients undergoing adjuvant chemotherapy [14]. Therefore, in the present study, we investigated the prognostic role of PLCγ1 in these early breast cancer patients stratified according to the St. Gallen criteria and to their menopausal status.

The identification of criteria for accurate prognostication of disease relapse is crucial for the selection of patient candidates for adjuvant therapy. Recognizing patients with high recurrence risk can potentially enhance their treatment outcomes, with the adoption of more aggressive treatments from an earlier stage of the disease, which might ultimately offer better overall survival. Conversely, low-risk patients can undergo less aggressive therapy, and therefore they can enjoy a better quality of life.

PLCγ1 is activated through its phosphorylation by tyrosine kinases in the cell. The major tyrosine kinases that have been shown to activate PLCγ1 in the cell belong to the growth factor receptor superfamily [11], which include the activated receptors for human epidermal growth factor (i.e., HER1/2), fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor and insulin-like growth factor. Thus, these tyrosine kinase receptors can phosphorylate PLCγ1 on its three tyrosine residues: Y771 and Y783 located between the X and Y catalytic domains, and Y1253 located near the COOH-terminus domain. Upon phosphorylation, PLCγ1 shows increased enzymatic activity, whereby the phosphorylation at Y783 has been described as required for PLCγ1 activation in vitro and in vivo [18‐20].

Although the phosphoinositide cycle operates classically at the plasma membrane level, a phosphoinositide cycle operates also within the nucleus [21]. We previously observed a selective nuclear positivity for PLCγ1-pY1253 and PLCγ1-pY783 in patients with early breast cancer, indicating that the nuclear signalling of these activated forms of PLCγ1 may have a specific tumorigenic role [14]. The nuclear PLCγ1 can contribute to mammary carcinogenesis through the modulation of key pathways, including the phosphoinositide 3-kinase (PI3K) nuclear activation [22], and by regulating the expression of cell cycle regulators such as cyclin D1 and cyclin-dependent kinase 4 expression, and the nuclear export of the Cdk inhibitor p27-kip1 [23]. Conversely, the down-regulation of PLCγ1 expression in breast cancer cells results in decreased lung metastasis formation in mice [12]. However, the mechanism/s by which PLCγ1 favours migration and metastatisation remain unclear. PLCγ1 has an essential role in cytoskeletal changes needed for the acquisition of the metastatic phenotype [11, 24], and dephosphorylation of PLCγ1 on residue Y783 inhibits PLCγ1 activation, thus blocking PLCγ1-activated rearrangement of the cytoskeleton, and cell migration [25]. PLCγ1 can contribute to metastatisation by direct [26] or indirect [12, 26] activation of RAC1, thus inducing migration-supporting cellular structures, such as lamellipodia and filopodia.

In the present study, we have shown that the prognostic role of activated PLCγ1 expression is limited to ER-positive, Luminal breast tumours. Indeed, by using validated antibodies for immunohistochemistry on paraffin sections, different PLCγ1 phosphorylation sites were associated with different prognosis for the Luminal-A and -B molecular subtypes. Of note, pY1253-PLCγ1^High, but not pY783-PLCγ1^High, was a significant independent prognostic factor for postmenopausal patients with Luminal B cancers (HR, 2.4). On the other hand, this was reversed in the hormonal pre/perimenopausal setting, where pY783-PLCγ1^High, but not pY1253-PLCγ1^High, was a particularly strong and significant risk factor for metastatic relapse (HR, 20.1).

Several multigene assays can now be included in clinical practice, such as the Oncotype DX, Prosigna and MammaPrint assays. Compared to the use of standard prognostic criteria, these multigene assays can provide some improvements in the recognition of patients with early stage ER-positive, HER2-negative breast cancer that will be at risk of recurrence. Indeed, at present, randomised controlled trials [27] are ongoing to prospectively validate their clinical usefulness. The future of diagnostic/ prognostic testing in ER-positive breast cancer is likely to rely on devising and reliably deploying assays that can predict the benefits of additional therapies, including newer targeted therapies. As no particular technology holds the key, immunohistochemistry remains a well settled, widely diffuse, and low-cost technique, and so it can have a role in the choice of adequate treatment [28].

Although the relationship between oestrogen stimulation and PLCγ expression has been explored in depth, recent data [29] have indicate a role for PLCγ1 in the proliferation of ER-positive tumour cells. Cells must increase chaperone levels to fold and sort proteins required for ERα-dependent cell proliferation. The unfolded protein response (UPR), which is an endoplasmic reticulum stress sensor, controls protein folding homeostasis. The UPR is overexpressed in several tumours where an early, pathological, activation of UPR occurs before the accumulation of unfolded proteins. In ERα-positive breast and ovary cancer cells, 17β-oestradiol induces rapid anticipatory activation of the UPR that is strictly PLCγ1 dependent. ER-positive breast cancers demonstrate elevated expression of a UPR gene signature that is also a prognostic marker associated to high risk of relapse and poor survival, and also resistance to tamoxifen therapy. Therefore, this PLCγ1-dependent anticipatory activation of the UPR defines a new role for oestrogens that can create a supportive environment for cancer cell proliferation and resistance to therapy, and might represent a new target in breast cancer [30]. Considering this aspect, it has also been reported that PLCγ1 activation downstream of FGFR-3 signalling is a critical event in the control of MAPK and PI3K activation, which can induce resistance to tamoxifen treatment [31].

Although the mechanisms involved remain to be defined, the activation of PLCγ1 as assessed by immunohistochemistry is a strong prognostic factor that can discriminate between high-risk and low-risk patients with hormone-receptor-positive early breast cancers. PLCγ1 might thus serve as a new target especially for treatment of Luminal-A pre/perimenopausal patients with T1-T2, N0 disease.