The Role of Beclin 1 and HER2 in Colorectal Carcinoma; An Immunohistochemical Study

Objective: This study aimed to evaluate the expression of Beclin 1 and HER2 proteins using immunohistochemistry in CRC tissues compared to colonic adenoma, and to investigate the correlation of their expression with clinicopathological parameters and survival outcomes in CRC patients. Methods: The study utilized paraffin-embedded blocks from 17 colonic adenoma and 81 CRC cases. Immunohistochemical analysis was performed to assess the expression of Beclin 1 and HER2 proteins. Results: The cytoplasmic expression of Beclin 1 was significantly higher in CRC tissues compared to adenoma specimens (P=0.051). High Beclin 1 expression was significantly associated with distal colon location (P=0.028). High HER2 cytoplasmic expression was significantly associated with vascular invasion (P=0.05), perineural invasion (P=0.03), and shorter overall survival (P=0.035). Conclusions: The findings suggest that Beclin 1 plays a role in colorectal carcinogenesis, with higher expression observed in CRC cases compared to adenoma cases. Furthermore, HER2 carries poor prognostic impact in CRC cases.


Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third most common cause of cancer-related death in both men and women worldwide with 1931590 newly diagnosed cases in 2020 and mortality rate of 935173 [1].In Egypt, cancer colon ranks the ninth among all tumors with 3430 newly diagnosed cases in 2020.Number of deaths due to cancer colon is 1910 with percent of 2.1 from all tumor's deaths in Egypt [1].
Despite advances in treatment options such as surgery, chemotherapy, and radiotherapy, the prognosis for CRC remains poor, particularly for patients with advanced disease as the five year survival rate reached 10-15% and about 40% of early staged cases experienced recurrence [2].Consequently, there is a pressing need to develop innovative therapeutic strategies for CRC, including targeted receptor therapy and immune-oncology approaches, as well as the identification of molecular biomarkers for early detection [3].
Autophagy, a cellular process involved in maintaining cellular homeostasis, has been implicated in both tumor promotion and suppression, depending on the context [4].Autophagy appears to be notably enhanced in colorectal cancer, and it has been linked to poor prognosis and drug resistance [5], however, autophagy studies in colorectal cancer show conflicting results, particularly in terms of

RESEARCH ARTICLE
The Role of Beclin 1 and HER2 in Colorectal Carcinoma; An Immunohistochemical Study chemotherapy resistance [4,6].
Beclin 1 is a critical regulator of autophagy and has been implicated in cancer development.However, its effects in cancer are still poorly understood.Studies have shown that Beclin 1 deficiency promotes tumor growth in breast cancer by regulating WNT1 [7], while others have demonstrated its requirement for carcinogenesis in breast cancer progenitor cells [8].Low expression of Beclin 1 has been associated with a malignant phenotype and poor prognosis in gastric cancer [9].These findings suggest that Beclin 1 may have autophagy-independent functions, highlighting its potential distinct role in cancer.
The significance of Beclin 1 in the development of colorectal cancer is still controversial.Although Beclin 1 knockdown has been shown to decrease epithelial-mesenchymal transition and reduce CRC cell invasiveness.[10], ectopic expression of Beclin 1 leads to growth retardation of the CRC cells [11].So, the ability of Beclin 1 in regulating cell cycle progression of colorectal cancer cells remains unknown.
HER2 pathway activation is an essential route of resistance for anti-epidermal growth factor receptor (EGFR) therapy, which is one of the most important treatments for a variety of cancers.The proto-oncogene HER2 belongs to the EGFR family and is also known as ERBB2 or HER2/neu.HER2-positive malignancies develop via a mechanism that is tightly linked to HER2 gene amplification on chromosome 17q.HER2 is a 185-kDa transmembrane receptor tyrosine kinase that promotes cell proliferation and opposes apoptosis by stimulating the RAS-and PI3K-AKT signaling pathways [12].
Gain-of-function mutations in HER2 have the potential to cause uncontrolled cell proliferation and division, angiogenesis stimulation, and tumor development [13].HER2 is expressed in several tissues including epithelial cells and mammary tissue, and hence its presence is strongly implicated in breast and stomach cancers.Tratuzumab, a monoclonal antibody (MAB), has been demonstrated to have an overall excellent prognosis in patients with HER2-positive breast cancer.Additionally, HER2 targeted therapy has been used more frequently for metastatic gastric cancer [14].
Although some studies suggest that HER2-positive CRC cases have a poor prognosis, some clinical trials targeting the HER2 pathway have yielded promising results, with dual HER2 blockade with MABs (trastuzumab with pertuzumab) or the combination of MABs with tyrosine kinase inhibitors (trastuzumab with lapatinib) inducing durable tumor response in approximately onethird of patients' refractory to standard systemic therapy [15].
Vega-Rubn-de-Celis et al. found that Beclin 1 and autophagy are likely inhibited by HER2, which likely contributes to HER2-mediated tumorigenesis of breast cancer.They also found that strategies to block HER2/ Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers [16].

Aim of the study
This study aimed to investigate the immunohistochemical expression of Beclin 1 and HER2 in colonic adenoma and CRC cases, and their correlation with clinicopathological parameters and survival outcomes.By elucidating the roles of Beclin 1 and HER2 in colorectal cancer, this research may contribute to the development of novel prognostic markers and targeted therapies for CRC patients.

Materials and Methods
This retrospective case control study was performed on 98 colonic surgical specimens obtained from Egyptian patients, including 81 cases of colorectal carcinoma (colectomy specimens) and 17 cases of colonic adenomas (colonoscopic biopsies).The specimens were collected between 2015 and 2019 from the Pathology Department at Menoufia University's Faculty of Medicine.The study was conducted in accordance with an approved Institutional Review Board (IRB) protocol.Data regarding clinical features and overall survival (OS) were extracted from patients' medical records.OS was calculated from the date of diagnosis to either the time of death or the last follow-up visit.Recurrence-free survival time was calculated from the date of surgery until the occurrence of a recurrence.Data regarding both of overall and recurrence free survival was available for only 54 cases.
*Construction of Tissue microarray (TMA) block: H&E stained slides from selected cases were carefully examined to identify viable and representative areas of each sample.A manual tissue arrayer's needle (Breecher Instrument, USA) was used to create tissue microarrays with a 2 mm punch size.Three cores from different areas of the tumor and stroma were sampled from each tumor specimen [21].

*Immunohistochemical staining
Two sections were cut from each TMA block and immunostained using the streptavidin-biotin-amplified system.The primary antibodies used were polyclonal rabbit anti-human antibodies, including Beclin 1 (concentrated with dilution of 1:100, Cat#ab114071) and HER2 (concentrated with dilution of 1:100, Cat#ab214275), purchased from Abcam (Cambridge, UK).

*Evaluation of immunohistochemical results
The expression of Beclin 1 was either cytoplasmic or nucleo-cytoplasmic.The immunohistochemical cytoplasmic staining was evaluated based on both the percentage of stained cells and the immunostaining intensity [22].
Pattern of HER2 expression: membranous/ cytoplasmic/membranous + cytoplasmic was evaluated.The immunohistochemical cytoplasmic staining was evaluated based on both the percentage of stained cells and the immunostaining intensity [23].
Furthermore, H-score was used for both Beclin 1 and HER2 and it was calculated by adding (1 x mildly stained cells) + ( 2 x moderately stained cells) + (3 x strongly stained cells) [24].cases were grouped using H-score into Low (≤median) and high (>median).

* Statistical analysis
Data was collected, tabulated, and analyzed using IBM (statistical package for the social sciences (SPSS) software package version 20 (Armonk, NY: IBM Corp).Fisher's exact (FE) and chi-square (χ 2 ) tests were used for evaluation of qualitative data while Mann-Whitney (U) test was used for evaluation of quantitative data.Kaplan Meier curve and log rank test were constructed for survival analysis.Results with (P ≤ 0.05) were considered as statistically significant [25].
positivity.Among the colorectal carcinoma cases, cytoplasmic expression of Beclin 1 was observed in 80.2% of cases, while nucleocytoplasmic expression was detected in only 22.2% of adenocarcinoma cases.Notably, a high H-score of Beclin 1 was found in 58% of cases, primarily in conventional adenocarcinoma tumor types with grade 2 tumor differentiation (Plate 1).
Regarding HER2 expression, the majority of adenoma cases (94.12%) demonstrated positive cytoplasmic expression, with 47.1% exhibiting a membranous + cytoplasmic staining pattern.In CRC cases, 96.3% displayed positive cytoplasmic expression of HER2, but only twenty-two cases (17%) showed a membranous +

Clinicopathologic data of adenoma and colorectal carcinoma cases
The current retrospective study was carried out on 17 adenoma cases and 81 colorectal adenocarcinoma cases; the characteristics of both adenoma and carcinoma cases were presented in Table 1 and 2.

Immunohistochemical expression of Beclin 1 & HER2 in adenoma and CRC biopsies
In the adenoma cases, all samples exhibited cytoplasmic expression of Beclin 1, without any nuclear

Comparison between adenoma and colorectal carcinoma cases regarding expression of Beclin 1 and HER2 expression
A higher median H score of Beclin 1 was observed in CRC specimens compared to adenoma specimens (P-value = 0.051).On the other hand, no statistical difference was found in the expression of HER2 between the two groups (Table 3).

The relationship between Beclin 1 and HER2 with the studied clinicopathologic parameters of CRC cases
A high Beclin 1 score showed a significant association with colorectal cancer located in the distal colon, followed by the rectum (P-value = 0.028).Additionally, there was a near-significant association between high Beclin 1 score and older age (P-value = 0.066), infiltrative tumor margin (P-value = 0.075), and advanced Dukes' stage (P-value = 0.097) (Table 4).Regarding HER2 expression, three cases showed negative expression and the correlation was done on the positive cases only which showed that high H score was significantly associated with vascular invasion (P-value = 0.05) and perineural invasion (P-value = 0.03) (Table 5).The impact of Beclin 1 and HER2 immuno-expression on overall and recurrence free survival of CRC patients Higher HER2 expression was found to be significantly associated with shorter overall survival in CRC patients (P = 0.035) (Figure 1).However, neither Beclin 1 nor HER2 showed a significant correlation with recurrencefree survival.

Correlation between immunohistochemical expressions of Beclin 1 and HER2 in CRC cases
Unfortunately, there was no significant association between Beclin 1 and HER2 expression in the studied CRC cases.

Discussion
This study aimed to investigate and analyze the expression of Beclin 1 and HER2 in colorectal cancer (CRC) in relation to various clinicopathological variables, including survival data.Our results revealed that the immunohistochemical expression of Beclin 1 was mainly cytoplasmic.However, it is worth noting that several studies have demonstrated both nuclear and cytoplasmic localization of Beclin-1.In a cohort study of CRCs, nearly half of the cases exhibited a significant nuclear Beclin-1 staining pattern [26].
Cytoplasmic expression of Beclin 1 was significantly higher in CRC cases compared to the adenoma group, which is consistent with previous studies [27].Zhang     and clinicopathologic parameters in the current study is consistent with other studies [27].This could be explained by the findings of Schmitz et al., who failed to demonstrate the prognostic value of Beclin 1 in the complete CRC cohort, particularly in the wild-type KRAS subgroup.However, in the mutated KRAS subgroup, increased nuclear Beclin-1 expression was significantly associated with decreased overall survival (OS).This suggests an alteration of the autophagy flux in the context of KRAS mutation [22].It is also possible that nuclear Beclin 1 has an autophagy-independent role in colorectal carcinogenesis, as it has been discovered that nuclear Beclin 1 can influence RB protein expression, regulating cell cycle and colorectal cancer cell proliferation [29].
On the other hand, Koustas et al. found that CRC patients with low expression levels of Beclin 1 protein had better overall survival than those with high expression levels [30].In contrast, Wu et al. reported that high protein expression of Beclin 1 was positively associated with prolonged survival, suggesting a potential tumor suppressor role [31].These discrepancies among studies may be attributed to variations in sample sizes.
In this study, we also examined the expression pattern of HER2 and found that out of the total 81 cases, 78 (96.3%) stained positive for HER2.All 78 positive cases showed a cytoplasmic staining pattern, while only 22 cases exhibited an additional membranous pattern.A similar finding was reported in a previous study, which observed cytoplasmic expression of HER2 in the majority (93%) of CRC cases, with only one case showing both membranous and cytoplasmic expression [23].
The variability in HER2 positivity in CRC and the diversity in expression patterns have been previously noted.The lack of a globally standardized protocol for reporting HER2 expression in CRC, as well as differences between studies conducted in different geographical regions with varying confounding factors such as lifestyle and diet, contribute to the inconsistency in reported data [32].
The distinction between membranous and cytoplasmic expression of HER2 is crucial in current monoclonal antibody (MAB) therapy for HER2-related breast cancer, as the cytoplasmic expression is not considered a clinically relevant target [3].The specific cause of HER2 cytoplasmic expression in CRC is unknown, but the overexpression of promoter-binding proteins, leading to increased HER2 synthesis, suggests its presence [33].Recent literature suggests that cytoplasmic HER2 expression in colorectal cancer may be related to survival prognosis, which provides a reason for optimism [34].
Our results indicated that a high H-score of HER2 expression was significantly correlated with poor prognostic factors such as vascular invasion (p=0.052),perineural invasion (p=0.031), and shorter overall survival (p=0.035).These findings are in agreement with Abdul Razzaq et al. and Hasan et al., who found that HER2 overexpression was associated with more aggressive colorectal cancer [35,36].
HER2 is a 185-kDa transmembrane receptor tyrosine kinase that promotes cell proliferation and inhibits apoptosis by activating the RAS-and PI3K-AKT signaling pathways.Gain-of-function mutations in HER2 can lead to uncontrolled cell growth, angiogenesis stimulation, and tumor development [13].However, contrary to our findings, Ezz El Din et al. observed no significant association between HER2 expression and different clinicopathological parameters or overall survival [37].
Previous studies have demonstrated the presence of a Beclin 1-HER2 complex on the cell surface of HER2expressing breast cancer cells.However,in our study, we did not find a significant correlation between Beclin 1 and HER2 expression.This discrepancy could be attributed to the differences in tissue type and localization of both markers in our study.
In conclusion, Beclin 1 plays a role in colorectal carcinogenesis, with higher expression observed in CRC compared to adenoma cases.HER2 expression carries poor prognostic implications in CRC and can be used as a prognostic indicator and a potential target for therapy in CRC patients.However, further research is needed to better understand the complex interactions and roles of Beclin 1 and HER2 in colorectal cancer.

Figure 1 .
Figure 1.High-HER2 CRC Cases (H-score ≥100) were Significantly Associated with Shorter Overall Survival than low-HER2 CRC Cases (P = 0.035) et al. found that Beclin 1 overexpression suppressed tumor growth of colon cancer cells in a xenograft model by inhibiting proliferation (assessed by Ki67 expression) and inducing apoptosis (assessed by TUNEL) [28].Furthermore, Beclin 1 did not show a significant Plate 2. Immunohistochemical Expression of HER2 in Adenoma and CRC Shows Positivity in the Cytoplasme.Mild expression of HER2 in colonic adenoma (A)(IHCx200).Mild expression of HER2 in conventional CRC (B) (IHCx200).Moderate expression of HER2 in CRC (C)(IHCx200).Strong expression of HER2 in CRC (D)(IHCx400).

Table 1 .
Clinco-Pathological Data of Colorectal Carcinoma Group

Table 1
SD, Standard deviation; n, Number of cases

Table 2 .
Clinico-Pathologic Data of Adenoma Group

Table 3 .
Beclin 1 and HER2 in Colorectal CarcinomaComparison between Adenoma and Carcinoma Groups According to Beclin 1 and HER2 Expression SD, Standard deviation; U, Mann Whitney test; χ 2 , Chi square test; n, number; p, p value for comparing between the two studied groups

Table 4 .
Relation between Beclin 1 H Score and Clinicopathological Data in Cancer Colon Group.(% from raw) SD, Standard deviation; U, Mann Whitney test; χ 2 , Chi square test; FE, Fisher Exact; n, number; p, p value for comparing between the two studied groups correlation with most clinicopathological parameters, except for tumor location, where it was higher in the distal colon and rectum.The regional distribution of CRC is important due to genetic factors, as well as regional diet and traditions that may affect CRC risk.The lack of significant association between Beclin 1 expression

Table 5 .
Relation between HER2 H score and Clinical Data in Cancer Colon Group.(% from raw) SD, Standard deviation; U, Mann Whitney test; χ 2 , Chi square test; FE, Fisher Exact; n, number; p, p value for comparing between the two studied groups