NILCO biomarkers in breast cancer from Chinese patients

Breast cancer is a heterogeneous disease with four major genetic-signature subtypes [1]. However, breast cancer can be broadly divided into two main groups: 1) Estrogen receptor positive (ER+) and triple negative breast cancer (TNBC: ER-, PR- and HER2-). The majority of breast cancers are ER+, respond to estrogens, and are commonly treated with anti-hormonal and HER2 (ErbB-2) targeted therapies. ER+ positive and HER2/neu+ breast cancer cells show suppressed Notch signaling, which is probably limited by the overwhelming proliferative and survival effects of ER and HER2-dependent pathways [2].

In contrast, TNBC is mostly dependent on growth factors [i.e., insulin, insulin-like growth factor-I (IGF-I) and adipokines] [3, 4]. This aggressive form of the disease accounts for 15% of all invasive breast cancers showing an acutely early onset. TNBC is associated with poor survival and resistance to common therapeutic treatments. This difficult-to-treat form of breast cancer shows a tendency to overcome drug effectiveness [5].

Notch signaling is a hallmark of breast cancer [6, 7]. The role of Notch in breast cancer development has long been studied, particularly relative to its effects on angiogenesis [8]. Notch expression correlates to poor prognosis and drug resistance of breast cancer patients [9, 10]. A particular feature of Notch signaling is its variable outcomes, which are dependent on cell and microenvironment types [7]. Ductal and lobular breast carcinomas show variable levels of Notch expression [2].

Notch signaling is a key mediator of proliferation, survival, and possibly malignant invasion of TNBC. These data suggest that TNBC is heavily dependent on Notch signaling [11]. In line with this notion, TNBC seems to differentially-activate Notch. Indeed, Notch1 and Notch4 are overexpressed in TNBC [12]. Moreover, in contrast to normal and ER+ breast cancer tissues, the activation of Notch in ER– breast cancer is linked to survivin upregulation (an apoptosis inhibitor and cell cycle regulator), which suggests ER- breast cancer cells are dependent on Notch-survivin signaling [13].

Recent data indicate that breast cancer development is likely related to lifestyle and the result of being overweight. Obesity is associated with more than 100,000 incidents of cancer in the United States every year, particularly cancers of the breast, colon, and endometrium. The specific molecular mechanisms involved in the development of obesity-related breast cancers are unknown. However, the general picture suggests that obesity-related breast cancer is the consequence of multi-factorial causes [14, 15]. Several molecules with altered patterns of expression in obesity are involved in breast cancer (i.e., insulin and IGF-1, and adipokines) [16]. Leptin, the major adipokine secreted by adipose tissue, is also produced by malignant cells, and linked to increased levels of Notch and survivin in breast cancer [17–19], and can affect tumor angiogenesis [20]. Leptin signaling can influence pro-angiogenic, inflammatory and mitogenic events in breast cancer [21–24].

We have previously unveiled a complex crosstalk between Notch, IL-1 and leptin (NILCO) in breast cancer cell lines, which could be essential for leptin-induced proliferation, inflammation and angiogenesis [17]. Moreover, a functional Notch-leptin axis is found in mouse carcinogenic-induced [18] and syngeneic breast cancer [19]. In these mouse models, leptin signaling induces both the expression and activation of Notch. However, it is unknown whether NILCO and its targets could correlate and/or are differentially-expressed in human TNBC, ER-, and ER+ breast cancer tissues.

We propose that specific associations between NILCO biomarkers occur in breast cancer, which may differ in TNBC. To this end, we examined the expression and cellular localization of NILCO, and targets, via immunohistochemistry in a commercial array of breast cancer biopsies from Chinese patients. Data were also analyzed in silico via Pathway Studio9 software (Ariadine Genomics, MD) [4]. Data analyses suggest that significant associations exist between NILCO and targets in breast cancer tissues. Higher levels of leptin and Notch1 were found in malignant compared to non-malignant tissues. TNBC showed lower levels of DLL4 and IL-1R tI compared to ER- and ER+ breast cancer. TNBC and their stromas showed differential cellular localization of Notch1, Notch4, JAG1, leptin and VEGFR2. Taken together, these results suggest that differential patterns of NILCO and targets are found in TNBC versus ER- and ER+ breast cancer. Present data support the idea for the potential use of NILCO and related molecules as biomarkers in breast cancer.

Notch signaling is a hallmark of breast cancer that is frequently identified as an indicator of poor prognosis and advanced disease. Therefore, Notch signaling is being targeted for breast cancer treatment [7, 27]. Additionally, increased leptin signaling has also been related to breast cancer growth, angiogenesis and poor outcomes [24]. Leptin increased the expression and activation of several members of the Notch family of proteins in breast cancer cells and derived tumors [17–19]. VEGF and VEGFR2 can be regulated by leptin-Notch crosstalk, which was also affected by IL-1 signaling. Therefore, Notch, IL-1 and leptin crosstalk outcome (NILCO) could be essential for the integration of leptin’s proangiogenic, pro-inflammatory and proliferative actions in breast cancer [17]. Leptin could also be involved in the development of drug resistance, metastasis and relapse of breast cancer, which are related to cancer stem cells [24, 28]. Furthermore, leptin transactivated and induced the expression of ER [29], EGFR [30], HER2 [31, 32] and IGF-1R [33] in breast cancer.

The abrogation of leptin signaling impaired the growth of tumors and expression of angiogenic biomarkers in human breast cancer xenografts [34, 35], and in mouse carcinogenic-induced [18] and syngeneic mammary tumors, which was more evident in obese contexts [19, 21]. Moreover, accumulated evidence from these pre-clinical studies in mice reinforces the idea that leptin-Notch crosstalk plays an important role in breast cancer. Nevertheless, whether NILCO and targets are differentially-expressed in human breast cancer tissues, in relation to ER, PR and HER2 as well as EGFR and AR statuses, is unknown.

Here we show that NILCO components (Notch1, Notch4, JAG1, DLL4, leptin, OB-R, IL-1R tI) and target molecules (VEGF and VEGFR2) were co-expressed in breast cancer tissues, irrespective of ER, PR and, HER2 statuses. Remarkably, TNBC shows a differential pattern of expression and cellular localization of NILCO. TNBC showed lower protein levels of IL-1R tI and DLL4, and fewer nuclei and cytoplasms were positive for Notch4 and JAG1. In contrast, more TNBC cytoplasms were positive for leptin. Moreover, TNBC stromas showed distinctive patterns of Notch1 and VEGFR2 immnoreactivities. Notch1 and Notch4 expression were lower, but VEGFR2 expression was higher in stromas from TNBC compared with ER- and ER+ breast cancer stromas.

Notch1 was found in the majority of breast cancer tissues evaluated. This data was in agreement with a previous report that showed 100% of Notch1 expression in TNBC [36]. Notch4 expression was previously found in 73% of TNBC cases (n=29) [36]. Present data show that fewer TNBCs were positive for Notch4 (58%) compared to ER- (75%) and ER+ (84%), but the differences were not significant. However, nuclear localization of Notch4 was significantly lower in TNBC malignant cells and stroma.

Present data further confirm previous findings showing that TNBC cells in culture (MDA-MB231) secreted more leptin (approximately four-fold) than ER+ breast cancer cells (MCF-7) [34]. Notch-induced transcriptional activity was not previously correlated with Notch receptor levels in breast cancer cell cultures, but Notch-induced gene transcription was highest in TNBC cells [2]. The global biological relevance of these findings is unclear. Nevertheless, present findings might indicate that TNBC could greatly depend on leptin’s actions, which could underline the role of NILCO in this breast cancer type.

Notch1 expression was also associated with the cell proliferation marker, Ki67. This marker was detected in approximately 60% of breast cancer independently of the expression of hormone receptors. It was previously reported that Ki67 is found in 90% of TNBC [12] and its expression correlates to Notch4, which is induced by Notch1 in breast cancer samples [2].

The transformation of normal breast epithelial cells by increased Notch signaling was previously linked to the repression of apoptosis in vitro[9]. Notch NICD1 interacted and mediated p53 inactivation through phosphorylation in vitro[37]. Additionally, it was also suggested that Notch signaling regulated apoptosis specifically caused by p53-induced expression of Puma and Noxa in vitro[6]. However, our present data suggest that p53 was not associated with the expression of NILCO and its targets, and is independent of hormonal receptor status. This data may also suggest that Notch-induced apoptosis in breast cancer in vivo may not always be p53 dependent.

TNBC and ER- breast cancers are not responsive to steroid hormones, but are highly aggressive tumors that respond to several other growth factor-related signals [5]. TNBC frequently show EGFR expression and resistance to EGFR drugs that could be driven by the Notch pathway [38]. In these cancers, Notch, leptin and OB-R could further contribute to tumor growth via increased the survival of breast cancer stem cells [24]. Indeed, the abrogation of Notch can negatively affect stem cells [10, 27]. Moreover, inhibition of OB-R significantly reduces the expression of several stem cell self-renewal transcription factors (NANOG, SOX2, and OCT4), and induces a mesenchymal-to-epithelial transition in TNBC cells [39]. Our present investigations show that EGFR expression was found in more ER- and TNBC (five-fold) than ER+ tumors. Additionally, EGFR was associated with higher expression of Notch1. Interestingly, leptin-induced activation of EGFR was suggested as a potential mechanism that promotes metastasis as well as invasion and, migration of breast cancer [33].

Obesity could affect breast carcinogenesis by autocrine and paracrine actions mediated by two major adipokines: leptin and adiponectin [24]. Obese breast cancer patients show poor prognosis, higher aggressiveness, and drug resistance [40–42]. Accumulated evidence suggests that obesity could induce Notch signaling. Indeed, an intact leptin-Notch axis could be involved in obesity-related breast cancer [18, 19]. However, diverse factors from adipose and other organs could also influence breast carcinogenesis and tumor growth. Therefore, more investigations are necessary to understand obesity-related breast cancer causes and mechanisms [43, 44]. All tissue samples used in this investigation were from Asiatic women (mean age of approximately 50 years). Obesity in China is currently a health problem. In 2002, the prevalence of obesity in China was relatively low (overweight prevalence at about 22.8% and for obesity, 7.1%) compared with Western countries, but the rapid increase in obesity is alarming [45]. Unfortunately, body weight and obesity data were not available for the breast cancer tissues used in this study.

For the first time, we are reporting on a comprehensive data analysis on protein levels of NILCO and targets in three major groups of breast cancer: TNBC, ER-, and ER+. TNBC showed distinctive patterns of expression and localization of NILCO, which suggests these molecules may be useful as markers for disease progression and aggressiveness. It is known that inhibition of Notch [9] and leptin signaling [17, 21, 34, 39] can revert the transformed phenotype of human breast cancer cell lines. Thus, treatments aimed to abrogate NILCO could provide the development of novel therapeutic interventions. More research is needed to establish the biomarker and potential therapeutic values of NILCO, and target expression in breast cancer, particularly in obese contexts.