CCL5 as a potential immunotherapeutic target in triple-negative breast cancer

Abstract

Breast cancer (BC) is a leading cause of mortality among women in the world. To date, a number of molecules have been established as disease status indicators and therapeutic targets. The best known among them are estrogen receptor-α (ER-α), progesterone receptor (PR) and HER-2/neu. About 15%-20% BC patients do not respond effectively to therapies targeting these classes of tumor-promoting factors. Thus, additional targets are strongly and urgently sought after in therapy for human BCs negative for ER, PR and HER-2, the so-called triple-negative BC (TNBC). Recent clinical work has revealed that CC chemokine ligand 5 (CCL5) is strongly associated with the progression of BC, particularly TNBC. How CCL5 contributes to the development of TNBC is not well understood. Experimental animal studies have begun to address the mechanistic issue. In this article, we will review the clinical and laboratory work in this area that has led to our own hypothesis that targeting CCL5 in TNBCs will have favorable therapeutic outcomes with minimal adverse impact on the general physiology.

Figure 1

Spontaneous ccl5 gene transcription in 4T1 cells. 4T1 tumor cells (10⁷) were electroporated with 10 µg murine ccl5 promoter–reporter construct (−979 to +8), or its NF-κB mutant (a 3-base-substitution at −81 to −79. Also see Figure 3 below), or the IL-12 p40 promoter construct, which is expressed in a highly cell-type specific manner. Each electroporated sample was split into three equal wells as triplicates. Cells were harvested 48 h later. Cell lysates were used for luciferase activity measurement using a luminometer. Total protein content in each sample was used to normalize the data variation due to cell number differences. Data represent triplicate reading for each sample with standard deviation. CCL5, CC chemokine ligand 5.

Figure 2

Spontaneous NF-κB-binding activity in 4T1 tumor cells. Nuclear extracts (NE) were isolated from 4T1 cells (4T) and primary mouse macrophages (M). Five µg of NE were mixed with the wild type (wt) or mutant (mt) NF-κB probe (their sequences presented with the critical core sequence underlined). Various competitors and NF-κB antibodies and control Ig were used to assess the binding specificity and selectivity. The complexes were resolved on a 6% native polyacrylamide gel.

Figure 3

Role of NF-κB in ccl5 gene transcription in 4T1 cells. 4T1 tumor cells (10⁷) were electroporated with the murine ccl5 promoter–reporter construct, together with an expression vector for NF-κB p65 (a), or an IκBα mutant (b), at increasing amounts in terms of molar ratios of reporter (R) to effector (E). Cells were harvested 48 h later. Cell lysates were used for luciferase activity measurement. Results are expressed as relative activity to that of ccl5 promoter alone without the effector (p65 or IκBα-M).

Figure 4

Role of AP-1 and CREB in ccl5 gene transcription in 4T1 cells 4T1 tumor cells (10⁷) were electroporated with the murine ccl5 promoter–reporter construct, together with an expression vectors for IκBα mutant, A-Fos, and A-CREB at the reporter∶effector molar ratio of 1∶1. Cells were harvested 48 h later. Cell lysates were used for luciferase activity measurement. Results are expressed as relative activity to that of ccl5 promoter alone without the effectors. CREB, cyclic AMP response element binding protein.