Neogenin expression is inversely associated with breast cancer grade in ex vivo

Breast cancer is the most common cancer in women in both developed and developing countries [1]. In China, breast cancer accounts for 14.2% of all malignant tumors in Chinese women, while the percentage is 26.4% in the USA [2]. Despite advancements in early detection and treatment of breast cancer, breast cancer is still the leading cause of cancer-related death among women in the world [3, 4]. Clinically, tumor stage, histological grade, and different tumor markers have been useful in evaluating and predicting breast cancer progression, treatment response, and prognosis [5]. Recently, researchers have tried to classify breast cancer based on the profile of differential gene expression to advance individualized treatment, and to help predict prognosis of the patients [6]. For example, breast cancer with estrogen receptor (ER) and progesterone receptor (PR) expression has been associated with sensitivity to endocrine therapy [7, 8], whereas human epidermal growth factor-2 (HER2)-overexpressed or HER-2-amplified breast cancer is resistant to endocrine therapy [9], but is more sensitive to trastuzumab [10, 11]. ER, PR and HER-2-negative breast cancer (that is, triple negative breast cancer) [12] has the worst prognosis among all subtypes of breast cancer. To date, early detection is still key for survival of patients. Thus, identification and evaluation of novel tumor markers could help with early detection of breast cancer or the development of novel therapeutic targets for treatment of breast cancer patients.

Towards this end, our study focused on the protein neogenin, which is closely related to the human tumor suppressor gene deleted in colorectal cancer (DCC) [12] and which plays a role in mammary morphogenesis [13]. Neogenin has been shown to be expressed in a wide range of tissues in vertebrates, especially in sites where cells actively proliferate and migrate. Neogenin ligands include netrins and the family of repulsive guidance molecules (RGM) [14‐16]. Neogenin has various functions dependent on its activation by different ligands; for example, after binding to netrin-1, neogenin can improve axon guidance by chemoattractively promoting cell migration and adhesion, whereas when binding RGM?, neogenin functions as a chemorepellant for cells [17]. Overall, the neogenin-ligand interaction can influence cell migration [17], tissue morphogenesis [18, 19], tumor growth [20, 21] and regulation of inflammation [22]. However, neogenin can induce apoptosis of certain types of cells when its ligands are absent. In the mammary gland, it has been shown that netrin-1-activated neogenin can stabilize multipotent progenitor cap cells during mammary gland morphogenesis [22, 23], while another study showed that neogenin expression was inversely associated with mammary gland tumorigenesis [23]. However, in the original gene cloning and screening study [13], there was no alteration in neogenin expression observed in more than 50 types of human cancer cell lines, including breast cancer. In this study, we measured expression of neogenin mRNA and protein in breast cancer and compared this to expression in distant non-cancerous tissues in order to establish whether this receptor is clinically associated with breast cancer.

To date, neogenin expression has been shown to be downregulated in a variety of human cancers such as glioblastoma [26], colon cancer [27], prostate cancer [28], and breast cancer [24]. In the original neogenin cloning and screening study, Meyerhardt and colleagues [13] reported no alteration in neogenin expression in more than 50 different human cancer cell lines, including breast cancer cell lines. Later, Lee and colleagues [23] measured expression of neogenin in breast cancer cell lines and in eight matched breast cancer and adjacent non-cancerous tissues using Western blot, and in only breast cancer tissues by using tissue array. They found that neogenin expression was downregulated in both breast cancer cell lines as well as cancerous tissues and concluded that neogenin expression was inversely correlated with mammary carcinogenicity. Our current data support this and other previously published data [24] in that we have found an association between neogenin expression and breast cancer grade. We observed no neogenin expression in higher histological grade breast cancer compared to lower grade tumors, which is consistent with a study of glioma [22] where neogenin expression was inversely associated with histological grade of that cancer. Neogenin expression has been reported to be even lower in recurrent glioma cases compared to that of their primary tumors [22]. The histological grading system in breast cancer is based on differentiation of tumor cells, which is an important factor in predicting prognosis of breast cancer patients and tumor aggressiveness. Thus, we speculate that breast cancer with lower neogenin expression in the high histological grade might be more likely to recur and/or have a worse prognosis. However, the majority of our patients had grade II and III breast cancers which precluded precise evaluation of neogenin expression in grade I breast cancer. Future studies should recruit patients with this breast cancer grade to further confirm that neogenin expression is associated with breast cancer grade.

In the current study, we observed that level of neogenin expression was particularly low in the four patients who were 65 years or older. In parallel with our findings, Bondy and colleagues [29] also showed that minimum levels of neogenin appeared in older glioma patients with poor prognosis. As older patients frequently have systemic diseases after being diagnosed with breast cancer, treatment of older patients is conservative compared to younger ones [30]. Thus, neogenin should be further evaluated as a potential biomarker for older breast cancer. Based on our clinical experiences, malignancy of breast cancer is closely associated with tumor size, subtype, TNM stage, lymph node metastasis, vascular invasion, and expression of other biomarkers (such as ER, PR HER-2, and Ki67); however, we failed to find any statistical significance between neogenin expression and these prognostic factors. This indicates that neogenin expression and its functions warrant further investigation in breast cancer. A previous study showed that the function of neogenin in normal breast development is to guide cap cells and luminal cells into juxtaposition during the adolescent development period [22]. Although the combination of netrin-1 and neogenin plays a significant role during this process, the role of neogenin in breast cancer remains to be determined. It has previously been shown that neogenin might function as an independent receptor in breast cancer to suppress tumor development [24]. When neogenin levels are downregulated, neogenin-induced apoptosis could be interrupted which could, in turn, lead to cancer development [20, 31]. Similarly, other studies suggest that neogenin could induce chemoattraction or chemorepulsion after binding to ligands such as netrin-1 and RGM to influence tumor cell migration and invasion [21, 32]. In addition, we have shown in the current study that neogenin expression is only marginally associated with lymph node metastasis of breast cancer.

As such, we hypothesize that the level of neogenin expression could be altered during tumorigenesis and that a lack of neogenin expression could promote tumorigenesis early in the process but that, following tumor formation, certain tumor cells may re-express neogenin protein to promote tumor cell migration and metastasis. However, further studies are needed to clarify the function of neogenin as well as the cause of lost neogenin expression in breast cancer before neogenin can be established as a biomarker for breast cancer diagnosis.

Written informed consent was obtained from the patient for the publication of this report and any accompanying images.