Roles of calreticulin in neuroblastoma

2.1 ER chaperones in cancer development

More and more evidences demonstrated that ER-resident chaperone proteins play critical roles in many pathological illnesses and a variety of human diseases [20].

Whether the involvement of ER-resident chaperone proteins is the primary cause or a secondary consequence in diseases is yet to be determined. Most findings of ER chaperones in human diseases have been focused on carcinogenesis and tumor progression. For example, our recent reviews showed that GRP78 is involved in cancer progression and drug resistance [4].

Moreover, the correlation between CRT expression levels and tumorigenesis has been extensively studied in various cancers and most reports have revealed that tumor tissues express significant higher levels of CRT compared to normal tissues [21].

Up-regulation of CRT presented in a variety of cancer cell lines, solid tumors, and human cancer cells, including oral cancer [22], breast ductal carcinoma [23, 24], colorectal cancer [25], prostate cancer [26], and vaginal carcinoma [27]. Tumor invasion, metastasis and recurrence were also positively correlated to increased CRT expression while the patients’ survival rate were inversely correlated to increased CRT expression in gastric cancer [28], breast cancer [29], bladder cancer [30], pancreatic cancer and esophageal squamous cell carcinoma [31, 32]. These results indicate that increased CRT expression might play a crucial role in cancer progression.

Another important role for CRT exposed on the cell surface, which is relevant for destruction of cancer cells, is via induction of the immune response [14, 33]. Results from several laboratories have demonstrated that cell-surface CRT facilitates the phagocytic uptake of apoptotic and cancer cells [34-36]. Obeid et al. demonstrated that drug treatments (anthracyclins) caused tumor cell to expose a surface pro-phagocytic protein, CRT, which induced immunogenic cell death [37]. It is becoming clear that surface exposure of CRT is required for phagocytosis on dying tumor cells. CRT expressed on the cell surface is considered as an “eat-me” signal and promote phagocytic uotake of cancer cells by immune system for multiple human cancers.

On the other hand, the roles of CRT in ovarian cancer progression are

inconclusive. Compared with primary tumors and solid metastases, reduced CRT expression was observed in malignant effusions of high-grade ovarian carcinoma along disease progression [38]. Besides, CRT expression levels may be associated with better response to chemotherapy while the survival was not related to CRT expression [38]. Very recently, mutations in calreticulin gene (CALR) were detected in a majority of myeloproliferative neoplasms [39-41]. Furthermore, in neuroblastoma, increased CRT expression is found to be associated with better prognosis and differentiated histologies [42, 43]. Therefore, the impact of CRT on tumor formation and progression may depend on different cell types and clinical stages.

2.2 Calreticulin in regulating cancer cell proliferation

Cancer formation and progression is characterized by rapid proliferation of mutant cells. Increasing evidence have revealed that manipulation of CRT expression had obvious effects on cell proliferation in various cancers. In pancreatic cells, overexpressed CRT enhanced cell growth, while knockdown of CRT had the opposite effect on cell growth [31]. In addition, reduction of CRT caused cell cycle arrest at the G0/G1 phase which resulted in significantly suppressed growth rate, colony-formation capacity, and anchorage-independent growth in oral cancer cell [22]. In bladder cancer, knockdown of CRT is also found to suppress cell growth

[15]. Although most studies have suggested that CRT expression positively regulates cell growth, other studies provided conflicting evidence on this issue. A recent study demonstrated that prostate cancer cells with higher CRT levels produced fewer colonies as well as inhibition of tumor growth both in vitro and in vivo [44].

According to these reports, whether CRT promotes or suppresses cell proliferation may rely on different cell types.

2.3 The biology of Neuroblastoma

Neuroblastoma (NB) is a common childhood tumor and the most frequently diagnosed malignancy in infancy, with more than 96% of patients diagnosed at the age of <10 years old [45, 46]. It is derived from the sympatho-adrenal lineage of embryonic neural crest cells [47]. Children with NB have a broad spectrum of clinical diversity that is highly associated with age at onset and genetic, biological, and pathological characteristics [47]. As a result, 50% of the NB patients die from this disease that continues to be one of the most challenging tasks among pediatric tumors.

The mechanism underlying its tumorigenesis remains obscure, despite the identification of several clinically relevant prognostic markers. Previous studies suggest that incomplete development and failure of differentiation or apoptosis of neuroblastic cells is critical in its development [48]. Previous studies have shown that

NB cells exhibit a capacity of differentiating into mature cells or spontaneous

regression by apoptosis [49, 50]. Studies also demonstrated that NB can be forced to differentiate upon the treatment of retinoic acid [51]. On the other hand, NB with better prognosis often express molecular markers indicative of cell differentiation, such as TrkA [52]. Furthermore, the expressions of apoptosis-related genes including p53, Bcl-2, and Bax have been demonstrated in NB and are correlated with favorable prognosis [53]. However, the factors contributing to the regulation of NB cell

differentiation or apoptosis are still unclear.

Genetic studies have postulated MYCN as one of the most important molecules that is closely related to the pathogenesis including differentiation, proliferation and apoptosis of NB. MYCN is a proto-oncogene normally expressed in the developing central and peripheral nervous systems [54]. Its expression is regulated by several signaling pathways, such as E2F1 [55]. As a bHLH transcription factor, MYCN dimerizes with MAX and binds to the E-box sequence (CACGTG) of the promotor region of target genes [56]. The major gene family that is directly regulated by MYCN is called the N-MYC Downstream Regulate Gene (NDRG) [57]. Through the transcriptional regulation of these genes, MYCN regulates the proliferation, growth, differentiation and survival of cells in the developing nervous system. In NB tumors, amplification of MYCN is closely associated with metastasis, advanced disease stages

and poor outcome [58].

2.4 ER chaperones and calreticulin in neuroblastoma

Though extensive exploration of ER chaperones in tumorigenesis, the roles of ER chaperones in neuroblastoma have not carefully addressed. Recently, Hsu et al.

have demonstrated that several chaperones including CRT, GRP75 and GRP78 are independent favorable prognostic markers in NB [42, 59, 60]. Hsu et al. found that the percentage of positive GRP78 immunostaining increased as the tumor histology of NB became differentiated (p=0.001) [60]. GTP78 expression as a significant factor for predicting favorable outcome has also been found in other studies of olfactory NB [61]. In addition, Hsu et al. have also found that positive GRP75 immunostaining is strongly correlated with differentiated histologies (p<0.001) and as an independent favorable prognostic factor [59].

Moreover, evidences also suggest that CRT may play an essential role in the biology of NB. Previous studies reveal that CRT is on the surface of NB cells and is essential for neurite formation when NB cells are induced to differentiate [62, 63]. Another in vitro study using NB cell line reveals that increased CRT expression is correlated with the differentiation of NB cells [64]. Recently, CRT has been identified by Hsu et al. as an independent favorable prognostic marker in

NB [42]. They have found that increased CRT expression in NB positively correlates with tumor differentiation and therefore predicts favorable outcome. In conclusion, CRT may play an essential role in the differentiation of NB cells, as well as in the differentiation of other neural progenitor cells or neural stem cells.

However, how CRT affects the differentiation of NB warrants further clarification.