**4. Calpain-1 as a potential prognostic factor in TNBC**

TNBC has been reported to have a clinical and pathological aggressive pattern due to its heterogeneous characteristic [44]. The ineffectiveness of hormonal and targeted therapies and poor prognosis for this subtype requires developing alternative therapeutic strategies such as biomarkers. The expression of a number of proteins has been shown to be associated with clinical outcome in TNBC patients [40, 45, 46]. Hence, there is a need to identify additional biomarkers to allow personalized treatment for patients with TNBC. For this reason, we explored the role of calpain-1 as a potential prognostic factor for TNBC therapy. We also evaluated the proliferation and apoptotic index for their potential use as possible prognostic factors since the biological behavior of tumor growth is a result of a balance between the proliferative activity and the number of cells dying by apoptosis [47]. Thus, they are considered as dominant histopathologic features in tumors. Several studies have also shown that calpain-1 expression significantly associated with tumor grade [40], proliferation [48, 49] and apoptosis [50]. Therefore, we also assessed the association between calpain-1 expression, cell proliferation and apoptosis in TNBC tissues.

#### **4.1. Patient characteristics**

We have shown in this study the effect of cisplatin on calpain-1 protein and its activation in TNBC cells. This has also been reported by others in other types of cancer cells [34, 35]. The finding that the increase in both calcium deposits and upregulation of endoplasmic reticulum

**Table 1.** Summary of results of experiments attempted to investigate the role of calpain-1 in the apoptotic death of TNBC

**Experiments Results**

Cisplatin (0 μm) + CPA

(50 μm)

Cisplatin (0 μm) + calpain-1 siRNA (150 nM)

Apoptosis was measured by Hoechst staining using fluorescent microscopy.

cells induced by cisplatin by way of the endoplasmic reticulum.

Cisplatin to induce endoplasmic reticulum stress (calcium release) and activate calpain-1 was assessed as activation of endoplasmic reticulum downstream effectors; α-fodrin and

152 Breast Cancer and Surgery

caspase-12. Calpain-1, α-fodrin and caspase-12 protein content (total and cleaved) was measured by Western blotting.

Cisplatin to activate calpain-1 by way of endoplasmic reticulum using CPA treatment was assessed as activation of endoplasmic reticulum downstream effectors; GRP78, calmodulin, α-fodrin and caspase-12, were measured using immunoblotting.

Cisplatin to activate calpain-1 by way of endoplasmic reticulum using siRNA treatment was assessed as activation of α-fodrin. The effect of calpain-1 siRNA on its content and activation (indicated by α-fodrin cleavage) was measured using immunoblotting

Control (μm/nM) Treatment after 24 hours P value of

Cisplatin (0 μm) Cisplatin (40 μm) P < 0.001 vs.

calpain-1 as reflected in cleavage of α-fodrin and caspase-12 and induced apoptosis in TNBC cells. Although cisplatin had no effect on calpain-1 content, it significantly caused cleavage of α-fodrin and caspase-12 and induced apoptosis in a dose-dependent manner [10].

CPA significantly enhanced upregulation of cisplatin-induced, calpain-1 activation and apoptosis compared with the controlled group

Calpain-1 small interfering RNA (siRNA) significantly attenuated cisplatininduced apoptosis in TNBC cells by downregulating calpain-1 in TNBC cells [10].

[10].

Cisplatin (0 μm) Cisplatin (20 μm) Cisplatin activated

Cisplatin (20 μm) + CPA

(50 μm)

Cisplatin

(150 nM)

(20 μm) + calpain-1 siRNA

apoptosis

control

P < 0.001 vs. control

P < 0.001 vs. CPA Control

P < 0.01 vs. Calpain-1 siRNA Control

> We tested calpain-1 protein expression and the proliferative/apoptotic index on paraffinembedded tissues from a cohort of 55 patients with TNBC. The main histological type was infiltrative ductal carcinoma in 96.4% (53 of 55), infiltrative lobular carcinoma in 1.8% (1 of 55) and micropapillary carcinoma 1.8% (1 of 55). Patients were females with a median age of 47 years (19–74). A total of 34 cases (61.8%) were premenopausal with no family history of breast cancer. Based on the disease indexing system, half (50.9%) of the patients were defined as stage III or IV at the time of diagnosis. Almost half of the patients ( = 26, 47.3%)

received neoadjuvant treatment and 5 (19.2%) achieved complete pathological response. Anthracyclines and taxanes were the most commonly used chemotherapeutic agents as frontline treatment. Breast cancer related overall survival (OS) was defined as the time interval (in months) from the date of diagnosis until death from breast cancer. Similarly, recurrence-free survival (RFS) was defined as the time interval (in months) between the start of primary treatment and date of cancer relapse.

created a diversion in the statistical analysis, (iii) the insufficiency of samples might have contributed to lack of significant correlations, (iv) the possibility of genetic differences between the populations in the current study and the ones already published may be the cause of differences on the expression of calpain-1 in breast cancer cells [40] and finally (v) the presence or absence of the hormonal receptors such as ER, PR, and HER2 that determine breast cancer behavior and thus treatment can influence the outcome. Storr *et al*. (2011) reported that there was no association between the expression of calpain-1 in HER2-positive breast cancer patients treated with trastuzumab following adjuvant chemotherapy with any of the clinicopathological variables [52]. Hence, their observation is consistent with our data but may differ

Triple-Negative Breast Cancer, Cisplatin and Calpain-1 http://dx.doi.org/10.5772/intechopen.74657 155

**4.4. Association between calpain-1 expression, cell proliferation and apoptosis in** 

Calpains have been reported to be involved in the proliferation of breast cancer cells [48, 49]. However, the role of the calpain family in proliferation of TNBC cells has not been reported yet. Ki-67, a nuclear antigen is a protein encoded by Ki-67 on 10q25 and considered to be a proliferation marker for predicting tumor development [53]. It is expressed during all active phases of the cell cycle except the resting phase, thus being present only in dividing cells. Ki-67 is detected by monoclonal antibody MIB-1 which can be a useful marker of proliferation and of prognostic value [53]. The quantitative assessment of Ki-67 staining on paraffin embedded tumor sections has been reported as an accurate estimate of the proliferation index of individual tumors [53].

Therefore, proliferative fractions of paraffin embedded breast cancer tissues were determined by immunohistochemical staining for Ki-67 antibody. The cellular proliferative activity was estimated as the percentage of tumor cells stained per field ×40. Statistical analysis showed no significant correlation between calpain-1 expression and proliferation ( = 0.29). Possible theories of the presence and absence of the hormonal receptors, differences in the genetic makeup, and other members of calpains involvement may also influence the correlation with proliferation.

Cell proliferation along with cell death are both phenomena responsible for control of cell number in normal tissues and tumors. Since chemotherapy induces programmed cell death by apoptosis, hence, the apoptotic tumor cells can be morphologically identified using the conventional hematoxylin and eosin (H&E) method and cells are counted using light microscopy. Therefore, there has been interest in the application of the apoptotic index in malignant growths as a putative prognostic marker. The percentage of apoptotic cells in tumor sections may also be measured by a molecular-based approach, labeling of fragmented DNA breaks and calculating the apoptotic index (AI) using the terminal transferase-uridyl nick-end labeling (TUNEL) assay. Therefore, in order to determine whether the frequency of apoptosis was related to tumorigenesis, two approaches; the conventional H&E staining method and the apoptotic TUNEL assay were both used to detect apoptotic cells and to prove that the two methods comparatively correlate with each other. H&E detects apoptosis in its degradation phase and can be subjective whereas the TUNEL assay detects apoptosis in its early phase and is more objective. Apoptotic cells were counted per 100 invasive tumor cells using ×40 objective. Apoptotic counts

in terms of the positivity of HER2.

**TNBC tissues**
