Clinical Significance of Notch Receptors In Triple Negative Breast Cancer

<jats:title>Abstract</jats:title>         <jats:p><jats:bold>Background: </jats:bold>The Notch signaling pathway is an evolutionary conserved cell signaling pathway that plays an indispensable role in essential developmental processes. Aberrant activation of Notch pathway is known to initiate wide array of diseases and cancers. The role of Notch receptors and the effect of their expression in Triple Negative Breast Cancer is unknown. <jats:bold>Methods and Results:</jats:bold> In this study, we evaluated the association between Notch receptors and clinicopathological parameters including disease-free survival and overall survival of one hundred TNBC patients by immunohistochemistry. We observed membrane, cytoplasmic and nuclear localization of Notch receptors in tumor cells of TNBC patients. It was observed that positive expression of nuclear Notch1 receptor (18%) was found be significantly correlated with high BR score (p=0.009) and necrosis (p=0.003) while cytoplasmic expression of Notch2 receptor (26%) was significantly correlated with local or distant metastasis (p=0.04), worse DFS (p=0.05) and poor OS (p=0.02) in TNBC patients. Further, it was determined that cytoplasmic (65%) and cytonuclear (3%) expression of Notch3 was significantly associated with poorly differentiated tumors (p=0.008) and necrosis (p=0.04) respectively. However, cytoplasmic Notch3 and Notch4 expression were negatively correlated with poor prognostic factors. <jats:bold>Conclusion:</jats:bold> Our data indicated that Notch receptors play a key role in promoting TNBC and mainly, Notch2 receptor may contribute to poor prognosis of the disease. Hence, it is implicated that Notch2 receptor may serve as a potential biomarker and therapeutic target for TNBC.</jats:p>

section_content: Breast cancer is the most commonly diagnosed type of cancer, accounting for 24. 5% newly registered cases in women and 11. 7% newly registered cases in both sexes in a year (https://gco. iarc. fr/today/data/factsheets/populations/900-world-fact-sheets. pdf). Globally, it is second leading cause of death in women after lung cancer (https://gco. iarc. fr/today/data/factsheets/populations/900-world-fact-sheets. pdf). Breast cancer is a phenotypically and biologically heterogeneous disease with diverse response to the treatments offered [1]. Immunohistochemical pro ling categorizes breast cancer into four molecular subtypes -luminal A, luminal B, Her2 enriched and Triple negative breast cancer [2]. Luminal A, luminal B and Her2 enriched subtypes are characterized by hormone receptor positivity and better prognosis than TNBC. Triple negative breast cancer is de ned by the lack of expression of hormone receptors (ER-, PR-, HER2-), which accounts for 15-20% of breast cancer cases [3, 4]. TNBC subtype has unfavorable prognosis and patients with TNBC are at greater risk of disease relapse or distant metastasis [5]. TNBC is characterized as an aggressive form of cancer which includes features such as higher tumor size, high histological grade, advanced stage and lymph node involvement [6]. Currently treatment available for TNBC comprises of surgery, traditional chemotherapy with anthracyclines, taxanes, platinum agents and radiotherapy [7]. 

Recently, Poly (ADP-Ribose) Polymerase (PARP) inhibitors are used as targeted therapy for TNBC patients with BRCA1/2 gene mutations and programmed death-ligand 1 (PD-L1) positive TNBC patients with locally advanced or metastatic disease are bene ted from PD-L1 inhibitors [8, 9]. Nonetheless, these therapies are bene cial to a small number of TNBC population. 

The Notch signaling pathway is a highly conserved pathway which regulates fundamental developmental processes such as cellular differentiation, proliferation, organ formation, morphogenesis and apoptosis [10]. The Notch signaling pathway is constitutively activated by the interaction of transmembrane Notch receptors and cell-surface ligands on adjacent cells [11]. Upon ligand activation, cascade of events is triggered which includes sequential cleavage by tumor necrosis factor alpha (TNF) converting enzyme (TACE) and gamma-secretase complex, releasing the Notch intracellular domain (NICD). The cleaved NICD translocates to the nucleus and binds with transcriptional regulators such as CBF-1/RBPjk, Suppressor of Hairless, and Lag-1 (CSL), Mastermind-like (MAML), p300 in order to initiate the expression Notch target genes [12, 13]. Abnormal expression of Notch receptors is known to play a crucial role in malignant transformation in haemopoietic malignancies as well as solid tumors [14]. Notch signaling pathway is responsible for normal development of mammary glands and the deregulation of Notch signaling pathway is involved in breast carcinogenesis [15]. Studies have frequently shown that Notch1, Notch3 and Notch4 are responsible for aggressiveness of TNBC by contributing to increased cell proliferation, anti-apoptotic activity and stem cell maintenance while few studies have reported tumor suppressive role of Notch2 receptors [16]. However, the role of Notch receptors in TNBC is quite debatable. The present study evaluated Notch1, Notch2, Notch3 and Notch4 expression by immunohistochemistry and their clinical signi cance in TNBC.
section_name: Introduction

section_name: Materials And Methods

section_content: One hundred female patients diagnosed with TNBC who had undergone surgery and treatment at the Gujarat Cancer & Research Institute, Ahmedabad during 2014 to 2019 were enrolled in this retrospective study. Detailed clinical history of the patients including age, menopausal status, tumor size, nodal involvement, AJCC TNM stage, histological type, histological grade and treatment offered was noted from the institutional records. Patients who had received chemotherapy or radiotherapy prior to the surgery were excluded from the study. Formalin xed para n embedded (FFPE) blocks were retrieved from archives of Histopathology Department of the institute. This study was approved by Scienti c Review and Ethics committees (EC/BHR/01/2020) of the institute.
section_name: Patients

section_content: Immunohistochemical analysis was used to detect the presence of Notch1, Notch2, Notch3 and Notch4 receptors in formalin xed para n embedded (FFPE) blocks of TNBC patients. 4µm sections from FFPE tumor tissue blocks were taken using a microtome (Leica Biosystems, Germany) and the sections were mounted on 3-Aminopropyl triethoxysilane (APES) coated slides. Immunohistochemistry was performed using autoimmunostainer instrument, Ventana Benchmark XT (Ventana Medical Biosystems, USA) and Ventana reagents according to the manufacturer's protocol. Brie y, the protocol includes depara nization with EZ prep solution, antigen retrieval using cell conditioning solution (CC1) at 95°C, followed by incubation with Ultra view DAB inhibitor for 4 minutes, 100µl of respective primary antibody for Notch1 (Monoclonal, 1:50, Invitrogen) for 32 minutes, Notch2 (Polyclonal, 1:25, Invitrogen) for 60 minutes, Notch3 (Polyclonal, 1:50, Invitrogen) for 60 minutes and Notch4 (Polyclonal, 1:150, Invitrogen) for 32 minutes, incubation with Ultra view Horseradish peroxidase (HRP) multimer for 8 minutes. The detection of antigen-antibody complex was carried out by 3,3'-diaminobenzidine (DAB), counterstaining using haematoxylin for 8 minutes and post counterstaining with bluing reagent for 4 minutes. Ultra view DAB detection kit was used for staining of Notch2, Notch3, Notch4 receptors and Opti view DAB detection kit was used for staining of Notch1. The protocol of Opti view DAB detection kit includes additional incubation with HQ linker for 8 minutes, counterstaining and post counterstaining for 16 minutes and 8 minutes respectively. The slides were later mounted using DPX and xylene.
section_name: Immunohistochemistry

section_content: Expression of Notch receptors (Notch1, Notch2, Notch3 and Notch4) was assessed by semiquantitative scoring method by employing Immunoreactive scoring (IRS) system. The percentage positive cells were graded as 0(negative staining), 1(<10% positive cells), 2(10% -50% positive cells), 3(51% -80% positive cells) and 4(>80% positive cells). Further, staining intensity of the cells was ranked as 0(negative), 1+ (weak), 2+(moderate) and 3+(strong). The immunoreactive score (IRS) for each case was obtained by multiplying the percentage of positive cells and the staining intensity which gives the range of 0-12. Median score was calculated and cases below the median score were considered as negative or low expression and cases above the median score were considered as over expression.
section_name: Evaluation of Immunohistochemistry

section_content: Statistical analysis was performed using SPSS 26. 0 (SPSS, Inc., Chicago, USA). The Chi-square test was to used determine the association between the expression of Notch receptors and clinicopathological parameters and the association between the Notch receptors. The correlation between the two parameters was determined by Pearson's correlation coe cient (r). Disease-free survival (DFS) time was calculated from date of diagnosis of the patient to date of disease relapse or date of last follow-up. 

Overall survival time was calculated from date of diagnosis of the patient to last follow-up or date of death due to cancer. Univariate survival analysis was performed by Kaplan-Meier method and signi cance of survival differences was calculated using log-rank test. Multivariate survival analysis was performed by Cox regression model with forward stepwise selection, Wald's statistics and hazard ratio (HR) with 95% con dence interval (CI) were calculated to estimate the prognostic signi cance. P value <0. 05 was interpreted as statistically signi cant.
section_name: Statistical Analysis

section_name: Results

section_content: Notch receptors were found to be localized in membrane, cytoplasm and nucleus of the tumor cells. As shown in gure1, positive expression of Notch1 was observed in 18 patients (18%), Notch2 in 31 patients (31%), Notch3 in 84 patients (84%) and Notch4 in 71 patients of TNBC (71%).
section_name: Correlation of Notch receptors with clinical and pathological parameters

section_content: Notch1 receptor was found to be predominantly localized only in the nucleus of the tumor cells of 18% patients as represented in Figure2. As shown in table 3, Notch1 nuclear positivity was signi cantly associated with high BR score (27%, 17/62, p=0. 009) and necrosis (31%, 13/42, p=0. 003) and a trend of association was observed between Notch1 positivity and high histological grade (23%, 15/66, p = 0. 08).
section_name: Notch1 receptor expression

section_content: Notch2 was localized in cytoplasm of tumor tissue of 26% patients and in nucleus of 4% patients. In 2% patients, coexistent cytoplasmic and nuclear expression of Notch2 was detected. The incidence of cytoplasmic Notch2 expression was signi cantly higher in patients with local or distant metastasis (47%, 7/15, p = 0. 04), while there was no signi cant association observed between nuclear and cytonuclear expression of Notch2 and clinicopathological parameters.
section_name: Notch2 receptor expression

section_content: It was observed that Notch3 receptor was found to be most frequently localized in the cytoplasm (65%), while 4% patients exhibited membrane expression, 13% patients and 3% patients exhibited cytomembrane and cytonuclear expression respectively. Although, membrane Notch3 expression was found in few patients, it was observed that tumors which were poorly differentiated exhibited high membrane Notch3 levels (23%, 15/62, p=0. 008). Also, a trend of association was observed between overexpression of membrane Notch3 and metastasis (33%, 05/15, p=0. 09). Contrary to this, trend of loss of Notch3 expression was observed in patients with high BR score (76%, 47/62, p=0. 06). Moreover, cytoplasmic Notch3 expression was more frequently detected in patients with well differentiated to moderately differentiated tumors (43%, 16/37, p=0. 01), low BR score tumors (52%, 14/27, p=0. 03), early TNM stage (100%, 02/02, p = 0. 05), lymph node negative status (51%, 29/57, p=0. 02) and perinodal extension negative status (44%, 35/79, p=0. 03). It was observed that cytonuclear Notch3 positivity was strongly correlated with necrosis (07%, 03/43, p=0. 04).
section_name: Notch3 receptor expression

section_content: Localization of Notch4 receptor was observed in cytoplasm of 54% cases, cytonuclear expression was observed in 13% cases and cytomembrane expression was observed in 4% cases. Low incidence of cytoplasmic Notch4 expression was detected in younger patients (72%, 36/50, p= 0. 02) and necrotic tumors (72%, 31/43, p=0. 04). However, cytomembrane and cytonuclear expression of Notch4 did not show any signi cant correlation with clinicopathological parameters. The association between Notch4 and clinicopathological parameters is listed in table8.
section_name: Notch4 receptor expression

section_content: It was observed that expression of Notch2 and Notch3 receptors was signi cantly correlated with expression of Notch4 receptor. Patients with increased levels of Notch2 expression exhibited increased levels of Notch4 receptor (84%, r=0. 19, p=0. 05) and similarly, higher levels of Notch4 receptor were detected in patients with positive Notch3 expression (76%, r=0. 26, p=0. 008). In addition, Notch1 expression was strongly correlated with nuclear expression of Notch3 (99%, r = 0. 22, p=0. 02). Further, it was seen that patients with increased levels of Notch1 receptor had decreased levels of cytoplasmic Notch3 receptor (95%, r=-0. 26, p=0. 007).
section_name: Intercorrelation of Notch receptors

section_content: The association between expression of Notch receptors and disease-free survival time and overall survival time of TNBC patients was investigated using Kaplan-Meier survival curves. The mean follow-up time was 37. 05 months and maximum follow-up time was 75 months. During this period, 15 (15%) patients had local or distant metastasis and 15 (15%) patients had died due to the disease. With regard to disease-free survival, patients expressing higher cytoplasmic Notch2 (27%, 7/26, 50. 71±4. 50 months) had worse disease-free survival than patients expressing lower cytoplasmic Notch2 (11%, 8/74, 66. 83±2. 71 months, χ 2 = 3. 77, df=1, p=0. 05). With regard to overall survival, it was observed that patients with higher incidence of cytoplasmic expression of Notch2 (31%, 8/26, 50. 60±3. 87 months) had poor overall survival than patients with low expression of cytoplasmic Notch2 (10%, 7/74, 67. 93±2. 48 months, χ 2 = 5. 44, df=1, p=0. 02). Expression of other Notch receptors did not show any statistically signi cant relationship with disease-free survival and overall survival.
section_name: Univariate Survival analysis for expression of Notch receptors in TNBC

section_content: Multivariate survival analysis using Cox regression model with forward selection method (likelihood ratio) revealed that, with regard to disease-free survival advanced TNM stage (Wald = 9. 36, HR = 0. 19, 95% CI = 0. 066-0. 551, p = 0. 002) entered at step 1 and high BR score (Wald = 7. 94, HR = 4. 93, 95% CI = 1. 626-14. 977, p = 0. 005) entered at step 2, indicating their role as poor prognostic factors. With respect to overall survival, greater tumor size (Wald = 10. 69, HR = 0. 57, 95% CI = 0. 10-0. 321, p = 0. 001) entered at step 1, positive lymph node status (Wald = 5. 482, HR = 7. 209, 95% CI = 1. 380-37. 676, p = 0. 019) entered at step 2, high BR score (Wald = 8. 355, HR = 7. 787, 95% CI = 1. 936-31. 317, p = 0. 004) entered at step 3 and higher grade (Wald = 8. 762, HR = 0. 012, 95% CI = 0. 001-0. 223, p = 0. 003) entered at step 4. 

Multivariate analysis carried out by Cox regression model to evaluate the prognostic role of Notch receptors expression, independent of clinicopathological parameters revealed that with respect to overall survival, cytoplasmic expression of Notch2 (Wald = 4. 87, HR = 3. 152, 95% CI = 1. 138-8. 734, p = 0. 027) entered at step 1.
section_name: Multivariate Survival analysis

section_content: Notch signaling pathway is known to play a vital role in normal development and growth. Notch signaling pathway is involved in embryonic development, cellular differentiation, proliferation, homeostasis and apoptosis. Numerous studies have reported that aberrant Notch expression is involved in neoplastic transformation and tumorigenesis. There are four known Notch paralogs which display different patterns in their expression and function across different malignancies. Oncogenic role of Notch signaling was rst reported in T-ALL [17] and later reported in lung cancer, ovarian cancer, breast cancer, colorectal cancer [18, 19, 20, 21, 22] while tumor suppressive role of Notch signaling was reported in pancreatic cancer and hepatocellular carcinoma [23, 24]. However, research about relationship between Notch receptors and TNBC, and the association among the Notch receptors is limited and unclear. 

In this study, we have examined the role of Notch receptors in TNBC cohort by analyzing the cellular localization of all four known Notch receptors by immunohistochemistry. We observed that Notch1 was expressed in the nucleus of tumor cells suggesting the translocation and accumulation of Notch intracellular domain (NICD1) in the nucleus of tumor cells. The accumulation of NICD1 in the nucleus was strongly correlated with high BR score and necrosis, indicating the tumor aggravating role of Notch1. Our data was in concordance with studies reporting the association of high grade tumors and poor prognosis of TNBC patients with up-regulated Notch1 [25, 26, 27]. 

Furthermore, upon examination of Notch2 protein it was revealed that Notch2 was localized in the cytoplasm and nucleus of tumor cells. Despite, various studies showing evidence of tumor suppressive role of Notch2 [28, 29, 30], our data suggests that Notch2 receptor plays a tumor promoting role in TNBC. It was observed that high cytoplasmic Notch2 expression was signi cantly correlated with local or distant metastasis, however nuclear and cytonuclear expression of Notch2 did not show any statistical signi cance with clinicopathological parameters. Nonetheless, our study was in line with evidence presented by Lee et al. and Sehrawat et al., stating that inhibition of Notch2 was responsible for antiapoptotic activity and inhibition of migration in TNBC cells [31, 32]. Also, our study demonstrated that high levels of cytoplasmic Notch2 was positively correlated with worse disease-free survival and poor overall survival proving its tumor promoting role. Earlier studies regarding Notch pathway emphasized that nuclear translocation of NICD was required in order to trigger the downstream target genes. Although, recently it has been demonstrated that in non-canonical Notch pathway, nuclear translocation is not necessary for the pathway to be activated and it may interact with other molecules in the cytoplasm to activate the pathway [33]. Therefore, it may be suggested that the association between cytoplasmic Notch2 expression and poor prognosis of the patients might be due to non-canonical Notch pathway. Moreover, our data represents that majority of TNBC samples exhibited high Notch3 protein levels in the cytoplasm. Along with cytoplasmic expression, membrane, cytomembrane and cytonuclear protein expression were also detected. Notably, it was revealed that up-regulated cytoplasmic Notch3 expression in tumors was associated with low BR score tumors, early TNM stage, lymph node negative status and perinodal extension negative status indicating that patients with up-regulated cytoplasmic Notch3 might have favorable prognosis. Our data was in line with studies demonstrating tumor suppressive role of Notch3. Zhang and colleagues observed that high expression of Notch3 inhibited cellular proliferation and tumorigenesis by activating PTEN resulting in better prognosis of breast cancer patients [34]. Another study by Chen and colleagues provided evidence that overexpression of Notch3 inhibited cell cycle progression by upregulating levels of Cdh1 in MDA-MB-231 cell lines. However, contrary to above ndings, it was observed that membrane and cytonuclear expression of Notch3 were strongly correlated with poorly differentiated tumors and necrosis respectively. Also, a trend of association was observed between frequent membranous Notch3 expression and metastasis. Although, cytonuclear expression of Notch3 was not detected frequently it was strongly associated with necrotic tumors suggesting the role of nuclear translocation in tumor promoting activity of Notch3. These results were parallel to the ndings of Choy and colleagues that revealed the involvement of nuclear Notch3 expression in tumor promotion [35]. The association of increased levels of Notch3 with poorly differentiated tumors might be due to the non-canonical pathway, where membrane-bound Notch3 may have interacted with cell regulators present in the cytoplasm. Contrary to that, membrane Notch3 expression was tended to associate with low BR score tumors. These results of Notch3 suggests that nuclear translocation of Notch3 is potentially involved in promoting tumor aggravating activity, whereas membrane-bound Notch3 may act as an oncogene via activation of non-canonical pathway which does not essentially require nuclear translocation. However, the strong association of high cytoplasmic Notch3 expression with low BR score tumors, early TNM stage, lymph node negative status and perinodal extension negative status might be due the immature state of the receptor or interaction of cytoplasmic Notch3 with tumor suppressing molecules which might be responsible for favorable prognosis of TNBC patients. 

In addition, in the majority of the patients expressing Notch4 receptor, it was frequently detected in the cytoplasm. This study revealed that high levels of cytoplasmic Notch4 were negatively associated with younger age and necrotic tumors. Contrary, to a study by Wang et al. which stated that Notch4 was associated with poor prognosis for TNBC patients [36], our data represents that Notch4 is inversely correlated to poor prognostic factors of the disease and may function as a tumor suppressor. Concurrently, few studies have presented over expression of Notch4 receptor as tumor suppressive in melanoma and endometrial cancer [37, 38]. 

Further, we evaluated the co-expression patterns of Notch receptors in TNBC patients. It was observed that Notch2 and Notch3 receptors showed positive correlation with Notch4 receptor. This may be interpreted as Notch receptors are signi cantly associated with each other and even though Notch4 might function as a good prognostic factor, together with Notch2 and Notch3 receptors they contribute to tumor promotion and poor survival of TNBC patients. Moreover, it was observed that nuclear expression of Notch3 was positively associated with nuclear expression of Notch1 suggesting the oncogenic role of nuclear localization of both Notch1 and Notch3 receptors. Additionally, it was noted that nuclear Notch1 expression was negatively correlated with the cytoplasmic Notch3 expression indicating opposing role of both the receptors based on their localization, as cytoplasmic Notch3 is associated with better prognosis and nuclear Notch1 is linked to poor outcome of the disease. Therefore, our results imply that interplay of Notch receptors is rather complex mechanism. 

In conclusion, our study demonstrated that Notch receptors exhibit a pleiotropic role in TNBC. Notch1 and Notch2 receptors play a tumor promoting role and contribute to poor outcome in TNBC patients. It was also revealed that although membrane and cytonuclear Notch3 had tumor aggravating effects, cytoplasmic Notch3 and Notch4 receptor had tumor suppressive effect. It is implicated that the tumor promoting activity of Notch receptors might be due to the canonical Notch signaling pathway which essentially requires the nuclear translocation of NICD to trigger the transcriptional regulation of target genes or it might be due to the non-canonical pathway which is activated through the interaction of Notch receptors with transcriptional regulators present in the cytoplasm to exert its effects. The tumor suppressive effect of cytoplasmic Notch3 and Notch4 expression may be due to the inactive state of the receptor or its interaction with tumor suppressive molecules in the cytoplasm. Therefore, it is important to assess the subcellular localization of the Notch receptors as they are ubiquitously distributed in the cells in order to evaluate their individual effects in TNBC. Hence, our results strongly indicate that Notch receptors are involved in promoting TNBC and chie y, Notch2 receptor may serve as a potential prognostic marker and therapeutic target for patients with TNBC.
section_name: Discussion

section_name: Statements And Declarations

section_content: Acknowledgments: This research was supported by The Gujarat Cancer Society and The Gujarat Cancer & Research Institute.

section_name: Funding

section_content: This work was supported by The Gujarat Cancer Society (GSC), Ahmedabad, Gujarat, India under grant number EC/BHR/01/2020.
section_name: Funding

section_content: The authors have no con ict of interest.
section_name: Con ict of Interest

section_name: Availability of Data and Material

section_content: Data and material of this study may not be available for another purposes.
section_name: Availability of Data and Material

section_name: Author Contributions

section_content: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ms. Heer Shah with the help of Ms. Mittal Mistry. The rst draft of the manuscript was written by Ms. Heer Shah under observation of Dr. Hemangini Vora and all authors commented on previous versions of the manuscript. All authors read and approved the nal manuscript.
section_name: Author Contributions

section_name: Ethics approval

section_content: Institute and The Gujarat Cancer Society (EC/BHR/01/2020), Ahmedabad, Gujarat, India on 23/03/2020.
section_name: This study was approved by Scienti c Review and Ethics committees of The Gujarat Cancer & Research

section_content: Informed consent was obtained from all individual participants included in the study.
section_name: Consent to participate

section_content: The authors a rm that all the participants in the study provided informed consent for publication of their individual data including medical history and treatment. 

Relationship between disease-free survival and Notch2

Relationship between overall survival and Notch2
section_name: Consent to publish