Cathepsin L is A Potential Marker for Triple-Negative Breast Cancer

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. C. Physiology & Molecular Biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidate important biological, chemical, or physical mechanisms of broad physiological significance. http://eajbsc.journals.ekb.eg/ Provided for non-commercial research and education use.


Introduction
Breast cancer is the second leading cause of cancer-related death among females worldwide (DeSantis, Ma et al. 2014).Approximately 1.7 million of diagnosed breast cancer resulted in corresponding 522,000 death (Tao, Shi et al. 2015).In Egypt, Breast cancer makes up 32% of all newly diagnosed cancer among females (Ibrahim, Khaled et al. 2014).Notably, breast cancer has different molecular subtypes based on the expression of the hormone receptors (HR), such as the estrogen receptor (ER) and progesterone receptor (PR), and the human epidermal growth factor receptor (HER2) (Li, Gonzalez-Angulo et al. 2011, Biswas, Efird et al. 2016).There are four main subtypes of breast cancer, which are defined as: luminal A (ER-positive and/or PR-positive and HER2-negative), luminal B (ER-positive and/or PR-positive and HER2-positive), HER2-positive (ER-negative, PR-negative and HER2-positive), and triple-negative (ER-negative, PR-negative and HER2-negative) (Lambertini, Santoro et al. 2016).
Triple-negative breast cancer (TNBC) represent 11.3% among Egyptian women with breast cancer (Salhia, Tapia et al. 2011).TNBC is the most aggressive subtype (Salhia, Tapia et al. 2011), because it presents with a higher histological grade and proliferation rate than other subtypes.It is associated with poor prognosis (earlier and higher risk of relapse and decreased overall survival) and shows a higher prevalence of germline BRCA mutation  Siemann 2015).There is one study showed increased levels of nuclear CTSL as a novel biomarker for subsets of TNBC patients (Grotsky, Gonzalez-Suarez et al. 2013).The aim of the present study was to determine the expression levels of CTSL mRNA in carcinoma tissues of TNBC vs. non-TNBC patients by qPCR.

MATERIAL AND METHODS Patients' Samples:
For patient recruitment, Institutional Review Board (IRB) approval was obtained from the ethics committee of Ain-Shams University, Cairo, Egypt.Dr. Mohamed Shinawi (co-author) enrolled 20 breast cancer patients from the breast clinic of Ain Shams university hospitals.According to molecular subtypes, breast cancer patients were divided into 2 subgroups; triple negative (TN) and non-triple negative (non-TN) patients.One part of the fresh carcinoma tissues was fixed in 10% PBS-formalin buffered solution for immunohistochemical staining and the other part was lysed for isolation of the total RNA.

Quantitative Real-Time PCR:
According to the manufacturer instructions, total RNA was isolated using GeneJET RNA Purification Kit (Thermo Fisher Scientific, USA) and 1 µg RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific,, CA, USA).Quantitative real-time PCR and melting curve analysis of CTSL mRNA expression gene was performed using SYBR™ Green PCR Master Mix (Applied Biosystems, USA) and StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA).Each sample was initially denatured at 95˚C for 10 min, then subjected to 40 cycles of the following: Denaturation at 95˚C for 15 sec, annealing at 55 ˚C for 30 sec and extension at 60˚C for 30 min.Melting curve analysis and 2% agarose gel electrophoresis of the PCR products was used to verify the product specificity.After normalization to GAPDH,, the 2 -∆∆Ct method was used to determine relative gene transcript levels (Livak and Schmittgen 2001).CTSL and GAPDH primers were purchased from Qiagen (Hilden, Germany).

Statistical Analysis:
Results were analyzed using SPSS (SPSS, Chicago, IL, USA), version 15.0.Differences among variables were evaluated using Fischer's exact tests.Mann-Whitney U-test (for non-normally distributed data) was used for two group comparisons.
Unless otherwise stated, all data are expressed as mean ± SEM.The level of significance was set at p < 0.05.The mRNA expression level of CTSL in carcinoma tissues of TNBC (n = 10) vs. non-TNBC (n = 10) patients was assessed using qRT-PCR.We found that there was a significant (P = 0.02) increase of CTSL mRNA expression by 7.2-fold in carcinoma tissues of TNBC than in non-TNBC patients (Fig. 1, and Fig. 2a & 2b).However, no significant correlation between CTSL expression and patients' clinicpathological data has been detected .

Fig.2a
Representative graph of the raw data analysis showing CTSL amplification plots of BC patients (n = 4).

Fig.2b
Representative graph of the raw data analysis showing CTSL melt curves of BC patients (n = 4).

DISCUSSION
In this study, we demonstrate a higher expression of CTSL mRNA level in carcinoma tissues of TNBC vs. non-TNBC.The high expression of CTSL has been assigned in different tumors whereby it modulates cancer invasiveness and metastasis (Mohamed and Sloane 2006).Therefore, CTSL may emerge as a biomarker and therapeutic target for TNBC.Our findings are in agreement with a study showed that nuclear CTSL is considered as a positive biomarker for TNBC (Grotsky, Gonzalez-Suarez et al. 2013) and CTSL level in serum may be a marker for invasion and metastasis in ovarian cancer (Sui, Shi et al. 2016).It is well established that TNBC is characterized by frequent somatic alterations of BRCA1 function and BRCA1-deficient cells activate CTSL through alterations in chromatin structure that either enhances the transcription of CTSL and/or alter the stability of CTSL mRNAs.This ultimately leads to bypass growth arrest via degradation of the DNA repair factor 53BP1 (Grotsky, Gonzalez-Suarez et al. 2013).The mesenchymal protein expression, namely Snail, vimentin and CTSL, was elevated in the TNBC compared to non-TNBC cells and that expression was functionally associated with the higher proliferative and migratory phenotype (Burton, Hawsawi et al. 2018).This observation was further confirmed in the clinical specimen of TNBC tissue but not in luminal A cancer tissue of African Americans as compared to Caucasian Americans patients (Burton, Hawsawi et al. 2018).Moreover, overexpression of Snail in the ER-positive MCF-7 cells resulted in enhanced migration and invasion via increased CTSL activity-mediated STAT-3 signaling (Burton, Smith et al. 2015).Of note, inhibition of nuclear localization of both CTSL and its substrate the transcription factor CCAATdisplacement protein/cut homeobox transcription factor (Cux1) was observed upon Snail and importin β1 silencing in MCF-7 cells (Burton, Henderson et al. 2017).Interestingly, treatment of the mesenchymal cells with the CTSL inhibitor Z-FY-CHO resulted in relocalization of CTSL from nucleus to cytoplasm, transition into epithelial cells, and decreased cell migration/invasion (Burton, Dougan et al. 2017, Burton, Henderson et al. 2017).In an immunohistochemical study of 188 breast cancer tissues, expression of CTSL has been shown to be associated with advanced cancer stages (Sun, Jiang et al. 2016).However, this correlation was not observed in our study.This could be attributed to the difference in characteristics of our collectives and to the low number of the enrolled patients.Overall, we suggest that the high expression of CTSL may act as a biomarker for TNBC patients and its targeting may represent a therapeutic strategy.However, our findings should be verified in a large group of breast cancer patients.

Fig. 1
Fig.1 Expression of CTSL mRNA in carcinoma tissues of TN BC vs. non-TN BC patients by using qPCR.The expression of CTSL was up-regulated in TN (n = 10) relative to non-TN patients (n = 10) (8.95 fold).** P < 0.01 as determined by Mann-Whitney U-test.

Table 1 .
Clinical and pathological data of IBC and non-IBC patients