Combined Evaluation of MAP1LC3B and SQSTM1 for Biological and Clinical Significance in Ductal Carcinoma of Breast Cancer

Breast cancer is the leading cause of cancer death in women worldwide. The microtubule-associated protein light chain 3B (MAP1LC3B) and adaptor sequestosome 1 (SQSTM1) are two major markers for autophagy. Increased protein levels of MAP1LC3B and SQSTM1 are considered to be causes of autophagy inhibition or activation in various types of cancers. However, the roles of MAP1LC3B and SQSTM1 in breast cancer are still not clear. Using a tissue microarray from 274 breast invasive ductal carcinoma (IDC) patients, we found that tumor tissues showed higher protein levels of MAP1LC3B and cytoplasmic SQSTM1 in comparison to those in adjacent normal tissues. Moreover, high levels of MAP1LC3B were associated with better survival, including disease-specific survival and disease-free survival (DFS) in IDC patients. Furthermore, high co-expression of MAP1LC3B and SQSTM1 was significantly associated with better DFS in IDC patients. Astonishingly, the autophagy inhibitor accumulated the protein levels of MAP1LC3B/SQSTM1 and enhanced the cytotoxic effects of cisplatin and paclitaxel in MCF7 and BT474 breast cancer cell lines, implying that autophagy inhibition might result in poor prognosis and chemosensitivity in IDC. Taken together, high co-expression of MAP1LC3B and SQSTM1 might serve as a potential diagnostic and prognostic biomarker for IDC patients.


Introduction
Breast cancer is the most prevalent type of malignancy among women and a major leading cause of cancer death worldwide. Breast invasive ductal carcinoma (IDC), a common type of breast cancer, comprises nearly 70-80% of all breast cancer [1]. Traditional classification systems in biological characteristics of molecular subtype specific proteins may have limitations for breast cancer patient-tailored treatment [2], suggesting the urgent need for effective diagnostic and prognostic biomarkers in breast cancer patients.
Autophagy is crucial for physiological homeostasis, but its dysfunction causes many diseases including cancer, neurodegenerative diseases and infection. Moreover, autophagy

Tissue Specimens
Margin-free (margin-size ≥ 0.2 cm) paraffin-embedded materials of CTAN and tumor tissues were obtained from 274 IDC patients. Clinicopathological data including age, pathological stage, TNM classification and cell differentiation were collected from patient records. TNM classification was determined at the time of initial tumor resection in accordance with the guidelines of the 2002 American Joint Committee. Written informed consent was not required as all data and specimens were previously collected and analyzed anonymously. This study was approved by the Institutional Review Board of the Kaohsiung Veterans General Hospital (VGHKS12-CT2-07) and was conducted following the Helsinki declaration.

Tissue Microarray (TMA) Construction
All paraffin-embedded tissues were used for TMA block construction. A TMA block consists of 48 trios; each trio contains one core with CTAN tissue and two cores with tumor tissue from each IDC patient. Each TMA block was cut into 4 µm-thick paraffin sections for further immunohistochemical staining [1].

IHC Scoring
A semi-quantitative approach based on staining intensity and percentage was used to score the degree of immune reactivity for IHC. The scores of staining intensity and percentage of the positive cells were added to obtain final scores ranging from 0 to 7. IHC scoring procedures were described in greater detail in our previous study [21].

Western Blotting
Cell lines pre-treated with bafilomycin A1 (BafA1, 100 nM, Selleckchem, Houston, TX, USA), concanamycin A (ConA, 10 nM, Merck & Co., Inc., Kenilworth, NJ, USA) and chloroquine (CQ, 20 µM, Sigma-Aldrich Corporation, St. Louis, MO, USA) were lysed with a RIPA buffer containing 0.1% SDS, 150 mM NaCl, 50 mM Tris-Cl pH7.5, 1% NP40, 0.25% sodium deoxycholate and a protease inhibitor cocktail. The different protein molecules in the cell lysates were separated by SDS-PAGE and then transferred to nitrocellulose membranes. The membrane was incubated with the primary antibodies at 4 • C overnight after blocking with 5% skim milk and then probed with HRP-labeled secondary antibody. Afterwards, the membrane was detected with ECL reagent, and the protein level was analyzed using the BioSpectrum ® Imaging System (UVP, Inc., Upland, CA, USA).

Live/Dead Cell Viability Assay
Cell lines (5 × 10 3 mL) were first pre-treated with autophagy inhibitors (20 µM CQ) for 5 h then cultured in Nano Culture Plates (NCPs; MBL Corporation, Ottawa, llinois, USA) for formation of cell spheroids. Afterwards, spheroid cells were treated with or without 50 µM CIS or 0.05 µM PTX for 24 h. Subsequently, the live/dead cell viability assay (LIVE/DEAD ® Viability/Cytotoxicity Kit, Thermo Fisher Scientific, Waltham, MA, USA) was used for analyzing the cell viability. The live (green)/dead (red) spheres were observed by fluorescence microscopy, and the cell viability was quantitated by a Fluoroskan Ascent FL reader [23].

Statistical Analysis
The different protein levels between CTAN and the tumor tissues were analyzed using the Wilcoxon signed-rank test. The correlation between protein levels and clinicopathologic parameters were evaluated using a Student's t test, Mann-Whitney U test, Kruskal-Wallis one-way ANOVA test and one-way ANOVA test. Disease-specific survival (DSS) was calculated as the date from the initial resection of the primary tumor to the date of cancerspecific death or last follow-up. Disease-free survival (DFS) was calculated as the date of the primary tumor resection to the date of recurrence or last follow-up. For survival analysis, the high and low levels of SQSTM1 and MAP1LC3B were dichotomized using cutoffs of 50%. A two-tailed p-value ≤ 0.05 was considered statistically significant.

Comparison of MAP1LC3B and SQSTM1 Expressions between CTAN and Tumor Tissues in IDC Patients
We analyzed the protein levels of MAP1LC3B and SQSTM1 in tissues of IDC patients by IHC staining. The scores of staining intensity for SQSTM1 and MAP1LC3B were measured using a numerical scale (−, no expression; +, weak expression; ++, moderate expression; and +++, strong expression, Figure 1A). We found that protein levels of MAP1LC3B (4.41 ± 2.18 vs. 5.14 ± 1.84, p = 0.001, Table 1) and SQSTM1 (0.51 ± 1.16 vs. 3.84 ± 2.24, p <0.001, Table 1) were higher in the tumor tissue than in the CTAN tissues of IDC patients ( Figure 1B,C). These results indicate that the levels of MAP1LC3B and SQSTM1 in tumor tissues were different from those in CTAN tissues of IDC patients.

Correlation of MAP1LC3B and SQSTM1 Expressions with Clinicopathological Outcomes and Prognosis in IDC Patients
To further investigate the clinical role of MAP1LC3B and SQSTM1 in IDC patients, the correlation of MAP1LC3B and SQSTM1 expression with clinicopathological outcomes in IDC breast patients was analyzed. We found that only high protein levels of MAP1LC3B were correlated with poor cell differentiation (p = 0.045, Table 2) in IDC patients. Moreover, we analyzed the correlation of MAP1LC3B and SQSTM1 expressions with DSS and DFS in IDC patients. After adjusting for AJCC pathological stage (II and III

Correlation of MAP1LC3B and SQSTM1 Expressions with Clinicopathological Outcomes and Prognosis in IDC Patients
To further investigate the clinical role of MAP1LC3B and SQSTM1 in IDC patients, the correlation of MAP1LC3B and SQSTM1 expression with clinicopathological outcomes in IDC breast patients was analyzed. We found that only high protein levels of MAP1LC3B were correlated with poor cell differentiation (p = 0.045,  Table 4). These results indicate that high co-expression of MAP1LC3B and SQSTM1 was correlated with better DFS in IDC patients.

Protein Levels of MAP1LC3B and SQSTM1 in Breast Cancer Cell Lines
To confirm that MAP1LC3B and SQSTM1 expressions were up-regulated due to autophagy inhibition, breast cancer cell lines (MCF7 and BT474) were treated with or without autophagy inhibitors [BafA1(100 nM), ConA (10 nM) and CQ (20µM)] for 5 h before harvesting. Accumulated protein levels of MAP1LC3B and SQSTM1 in treated breast cancer cell lines were observed ( Figure 2A) and quantified ( Figure 2B). These results indicate that the accumulation of MAP1LC3B and SQSTM1 might be caused by autophagy inhibition in breast cancer cell lines.
To confirm that MAP1LC3B and SQSTM1 expressions were up-regulated due to autophagy inhibition, breast cancer cell lines (MCF7 and BT474) were treated with or without autophagy inhibitors [BafA1(100 nM), ConA (10 nM) and CQ (20μM)]for 5 h before harvesting. Accumulated protein levels of MAP1LC3B and SQSTM1 in treated breast cancer cell lines were observed ( Figure 2A) and quantified ( Figure 2B). These results indicate that the accumulation of MAP1LC3B and SQSTM1 might be caused by autophagy inhibition in breast cancer cell lines.

Effect of Autophagy Inhibition on Chemoresistance in Breast Cancer Cell Line
In the study, higher protein levels of MAP1LC3B and SQSTM1 were found in tumor tissues, and the high co-expression of MAP1LC3B and SQSTM1 was associated with tumorigenesis and better DFS. Moreover, we found that MAP1LC3B and SQSTM1 might be accumulated due to autophagy inhibition in breast cancer cell lines. To determine the effects of autophagy inhibition on chemoresistance in IDC, a tumorsphere culture model and Live/Dead assay for drug resistance were used. Interestingly, MCF7 ( Figure 3A) and BT474 ( Figure 3B) cell lines pre-treated with autophagy inhibitor CQ showed decreased tumorsphere formation and enhanced chemosensitivity to CIS and PTX in a dose-independent manner ( Figure S1). Moreover, the Live/Dead staining assay ( Figure 4A) indicated that autophagy inhibitor CQ slightly reduced live MCF7 and BT474 cells ( Figure 4B) but significantly increased dead MCF7 and BT474 cells ( Figure 4C) in the presence of CIS and PTX. Taken together, these results indicate that accumulated MAP1LC3B and SQSTM1 caused by autophagy inhibition might be involved in the chemoresistance of breast cancer cell lines.
tumorsphere formation and enhanced chemosensitivity to CIS and PTX in a dose-independent manner ( Figure S1). Moreover, the Live/Dead staining assay ( Figure 4A) indicated that autophagy inhibitor CQ slightly reduced live MCF7 and BT474 cells ( Figure 4B) but significantly increased dead MCF7 and BT474 cells ( Figure 4C) in the presence of CIS and PTX. Taken together, these results indicate that accumulated MAP1LC3B and SQSTM1 caused by autophagy inhibition might be involved in the chemoresistance of breast cancer cell lines.  The sphere morphology and cell viability were detected under light microscopy (100× magnification) and using the CellTiterGlo 3D system, respectively Results represent the mean of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.
Biomedicines 2021, 9, x FOR PEER REVIEW cultured in a round bottom ultra-low plate for tumorsphere formation. The tumorspheres were then treated with or with out cisplatin (CIS, 50 μM) or paclitaxel (PTX, 0.05 μM) for 24 h. The sphere morphology and cell viability were detected under light microscopy (100× magnification) and using the CellTiterGlo 3D system, respectively Results represent th mean of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Discussion
MAP1LC3B and SQSTM1 are essential to the autophagy machinery. The accum tion of MAP1LC3B and SQSTM1 plays a different role in clinicopathological outc and prognosis in various types of cancer [3]. However, the biological role and clinica nificance of MAP1LC3B and SQSTM1 in breast cancer remains unclear. We found protein levels of MAP1LC3B and SQSTM1 were higher in tumor tissues than in C tissues in IDC patients. The high co-expression of MAP1LC3B and SQSTM1 was a ated with better DFS survival in IDC patients. The accumulated MAP1LC3B and SQS caused by autophagy inhibition increased chemosensitivity to cancer drugs (CIS and

Discussion
MAP1LC3B and SQSTM1 are essential to the autophagy machinery. The accumulation of MAP1LC3B and SQSTM1 plays a different role in clinicopathological outcomes and prognosis in various types of cancer [3]. However, the biological role and clinical significance of MAP1LC3B and SQSTM1 in breast cancer remains unclear. We found that protein levels of MAP1LC3B and SQSTM1 were higher in tumor tissues than in CTAN tissues in IDC patients. The high co-expression of MAP1LC3B and SQSTM1 was associated with better DFS survival in IDC patients. The accumulated MAP1LC3B and SQSTM1 caused by autophagy inhibition increased chemosensitivity to cancer drugs (CIS and PTX) in breast cancer cell lines. We showed that the accumulation of MAP1LC3B and SQSTM1 caused by autophagy inhibition was associated with better survival of IDC patients, providing a potentially novel therapy for IDC.
Autophagy functions are a "double-edged sword" for cancer, showing opposite functions in cancer initiation by limiting genome damage/mutation or promoting cancer-cell survival in stressed microenvironments [24]. An increasing number of studies have found that autophagy defects also result in tumor formation. Moreover, the expression of the intact autophagy genes is found to be downregulated in cancers, and the spontaneous frequency of cancer malignancies is increased due to an autophagy-related gene deficiency. For example, Beclin1 is an essential autophagy gene, and the Beclin1 +/− genotype in vivo produces spontaneous tumors such as lung and liver tumors and mammary hyperplasia [25]. Mono-allelic deletion of beclin1 has been found in many cancers such as breast, ovarian and prostate cancers [26]. The overall survival rate of patients with BECN1-negative tumors is significantly lower compared to patients with BECN1positive tumors [27]. Furthermore, the homozygote deletion of Atg5 increases predisposition to liver tumors within the high-penetrance mouse model [28]. Deregulating autophagy by the frameshift mutations of Atg2B, Atg5, Atg9B and Atg12 is involved in cancer development [29]. Somatic point mutations of Atg5 have been found in patients with gastric cancer, colorectal cancer and hepatocellular carcinoma [30]. These results suggest that autophagy defects might promote tumor formation.
Expressions of MAP1LC3B and SQSTM1 have been widely used to evaluate autophagy status in mammalian cells and their clinical significance in cancer patients. High MAP1LC3B expression is correlated with poor survival of patients with gastric cancer [31], OSCC [3] and other disease [8]. In contrast, cancer patients with high MAP1LC3B expression have better outcomes, such as in early-stage non-small cell lung cancer (NSCLC) [32]. Moreover, high MAP1LC3B levels are associated with reduced tumor aggressiveness in early-stage NSCLC [32]. Additionally, patients with low MAP1LC3B level have a significantly poorer prognosis than patients with high MAP1LC3B [27]. On the other hand, the accumulation of SQSTM1 is used as a marker for autophagy inhibition/defects [33]. The aberrant accumulation of SQSTM1 has been found in gastrointestinal cancer [34], prostate cancer [35], hepatocellular carcinoma [36], breast cancer [37] and lung adenocarcinoma [14], suggesting that SQSTM1 accumulation through autophagy inhibition correlates with cancer progression. In this study, we found that MAP1LC3B and SQSTM1 accumulated in IDC patients and IDC patients with the high co-expression of MAP1LC3B and SQSTM1 had better DFS, which is possibly due to these tumors becoming resistant to chemotherapy or radiation therapy [38]. Moreover, autophagy is known to be involved in chemoresistance [39]. These results implied that autophagy defects might be associated with IDC progression.
Although autophagy defects result in tumor formation, mosaic deletion of Atg5 in mice or Atg7 in mouse livers produces only benign hepatomas, suggesting that complete and specific autophagy deficiency promotes liver tumor initiation but restricts progression to malignant disease [28]. Similarly, loss of Atg5 or Atg7 in the mouse pancreas promotes benign pancreatic intraepithelial neoplasia (PanIN) formation but also prevents progression of this PanIN to malignant disease [25]. Moreover, SQSTM1 accumulation by autophagy inactivation contributes to the development of benign hepatomas in mouse models, but the underlying mechanism is not known [28]. Furthermore, autophagy defects could increase drug sensitivity for cancer therapy in different types of cancers [40]. We found that chemosensitivity increases in breast cancer cell lines treated with autophagy inhibitors. Moreover, IDC patients with autophagy defects were more likely to survive. These findings demonstrate that autophagy defects cause benign breast tumor formation but prevent poor prognosis in breast cancer patients.
Breast cancer is composed of multiple molecular subtypes such asER, PR andHER2, while triple-negative breast cancer (TNBC) lacks ER, PR and HER2 expression, leading to distinct morphologies, treatment responses and clinical outcomes [41]. Autophagy dependence in breast cancer may be subtype-dependent [26]. For example, HER2 positive tumors exhibit low levels of autophagy [42], showing low expression of Beclin1 and autophagy-related genes [43]. HER2 amplification is significantly associated with decreased BECN1expression in breast cancer tumors [44]. However, TNBC tumors exhibit a higher level of autophagy with high BECN1and MAP1LC3B expressions [45]. Thus, autophagy dependence according to molecular subtype in IDC patients requires further investigation.
The present study is subject to certain limitations. Our cell culture model indicates that autophagy inhibition sensitizes breast cancer cells to chemotherapeutic drugs, leading to better survival in IDC patients. Nevertheless, autophagic flux can be measured only in cell cultures and animal models [46], not in clinical patients [4,47]. Moreover, lysosomal enzyme defects may result in pseudo-autophagy [48]. Therefore, the possibility of lysosomal dysfunction for pseudo-autophagy in breast cancer should be further studied. Additionally, our results indicated that accumulated MAP1LC3B and SQSTM1 could be caused by defective autophagy and the autophagic defect was associated with better survival and chemosensitivity. Autophagy receptor MAP1LC3B and adaptor SQSTM1 play an important role in the recruitment of cellular components into autophagosome for bulk degradation. Therefore, the knockdown of MAP1LC3B or/and SQSTM1 might result in a defective autophagy, thus protecting cancer cells. However, MAP1LC3B is one of the autophagy receptors (such as MAP1LC3A, MAP1LC3C, GABARAP and so on) and SQSTM1 is one of the adaptors (such as NBR1, NDP52, TAX1BP1 and so on) necessary for autophagy function according to previous studies [49]. The other orthologous would compensate their function while silencing MAP1LC3B or/and SQSTM1 in cancer cells. Thus, the single knockdown of MAP1LC3B and SQSTM1 or combined knockdown of MAP1LC3B and SQSTM1 might have no significant effect on the cell function. Furthermore, the analyzed data indicated that poorly differentiated tumors have a worse prognosis in IDC patients. However, MAP1LC3B upregulation was associated with poor cell differentiation but better survival. The possible reason might be that the upregulation of lipidated MAP1LC3B alone in patients could be caused by either autophagy inhibition or induction. Thus, observing MAP3LC3B protein levels alone, it was hard to tell the association of autophagy induction or inhibition with poor differentiation. To more precisely evaluate the correlation of autophagy inhibition with prognosis, elevated protein levels of both MAP1LC3B and SQSTM1 were considered as autophagy inhibition. Our results showed that the high co-expression of MAP1LC3B and SQSTM1 as defective autophagy led to better survival in IDC patients.

Conclusions
Our results find higher protein levels of MAP1LC3B and cytoplasmic SQSTM1 in the tumor tissues of IDC patients. Moreover, IDC patients with high co-expression of MAP1LC3B and SQSTM1 had better DFS. The accumulation of MAP1LC3B and SQSTM1 resulting from autophagy defects could facilitate chemosensitivity in breast cancer cell lines. Thus, the high co-expression of MAP1LC3B and SQSTM1 might be a potential biomarker for future diagnosis and prognosis in IDC patients.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/biomedicines9111514/s1, Figure S1: Effects of autophagy inhibition on sphere cell viability in breast cancer cell lines treating cancer drugs.