Introduction
Breast cancer (BCa) remains the most common malignancy in women other than skin cancer, representing approximately one third of all malignancies diagnosed among women in the USA [
1,
2]. Since 2008, the incidence of breast cancer worldwide has increased by more than 20%, and mortality has increased by 14%. In order to treat breast cancer more precisely, several genetic drivers of breast cancer have been identified, and subclassification has been conducted based on either the coding or non-coding genome. The classification of breast cancer based on the coding region identified potential genetic targets including the
CCND1 gene, which is amplified in 30 to 58% of breast cancers; the estrogen receptor (ERα); and/or progesterone receptor (PR) and Her2. Because evidence suggests both the coding and non-coding genome may contribute to the onset and progression of tumorigenesis [
3,
4], subtypes of breast cancer have been identified using patterns of expression for both the coding [
5] and non-coding genomes [
6‐
8].
Using the coding genome, five distinct molecular subtypes were identified referred to as luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, basal-like, and claudin-low and normal-like [
9]. Triple-negative breast cancer (TNBC), which lacks ERα, PR, and Her2, is a deadly form of breast cancer. In 10 to 15% of cases, TNBC is associated with DNA damage repair protein mutations (
BRCA1 BARD1,
BRCA1,
BRCA2,
PALB2, and
RAD51D) [
10], in 19% with PD-L1 expression [
11], and in > 95% with CCR5 overexpression [
12].
Well known as an essential co-receptor for HIV, more recently, CCR5 has become strongly implicated in the progression of human cancer, in particular, metastatic cancer [
13]. CCR5, a seven trans-membrane G-protein coupled receptor (GPCR), is normally expressed only in the immune system; however, CCR5 becomes overexpressed in several malignancies and is overexpressed in breast cancer [
12,
13]. In the analysis of > 2200 breast cancer patients, > 50% of patient’s tumors were CCR5
+. and > 95% of triple-negative breast cancer (TNBC) were CCR5
+ [
12]. Several characteristics of CCR5 suggest the receptor may be important in human breast cancer. CCR5 receptor levels correlate with poor prognosis in breast cancer [
13‐
15]. CCR5 expression correlates well with increased tumor heterogeneity in breast cancer [
16,
17]. Upon transformation of breast epithelial cells, the increased expression of CCR5 results in increased motility and homing behavior to metastatic sites [
12,
13]. Furthermore, CCR5
+ breast cancer epithelial cells have both enhanced tumor-initiating capacity and form mammospheres with greater efficiency in mice [
13], a feature of cancer stem cells. Finally, ectopic CCR5 expression within cancer epithelial cells is sufficient to drive cancer cell metastasis [
12].
Several CCR5 antagonists developed for HIV treatment, including the small molecule CCR5 inhibitors (maraviroc and vicriviroc) and the humanized monoclonal anti-CCR5 antibody leronlimab, are currently being retasked for cancer and cancer-related diseases [
17,
18]. In HIV treatment, the small-molecule inhibitor maraviroc and the humanized monoclonal antibody leronlimab achieved their primary endpoints in phase 3 HIV clinical trials [
19‐
21]. CCR5-specific small molecular inhibitors prevented metastasis of isogenic oncogene-transformed breast cancer cells in NOD/SCID mice [
12] and prostate cancer metastasis in immune-competent mice [
22]. Unfortunately, maraviroc carries a “black box” warning due to the associated serious adverse including hepatotoxicity.
Leronlimab is an inhibitor of CCR5 signaling in immune cells. Currently, more than 800 patients with HIV have received leronlimab without serious adverse events related to the agent. Given the safety profile of leronlimab, and potential adverse events with the small molecular inhibitors, we conducted studies to determine whether leronlimab could bind and block CCR5 signaling in human breast cancer cells. These studies extend prior studies by showing CCR5 inhibition both prevents metastasis and reduces the progression of established metastasis in vivo.
Discussion
CCR5 abundance is induced by transformation of immortalized human breast cells with diverse oncogenes including Ha-Ras, c-Myc, ErbB2, and c-Src [
12]. Furthermore, DNA damage induced by radiation and chemotherapy is associated with increased expression of CCR5 within human breast cancer cell lines [
13]. Increased cytoplasmic CCR5 abundance correlates with poor prognosis in a variety of cancers including breast cancer [
13], gastric adenocarcinoma, and other malignancies [
18]. The rationale for the current studies includes evidence that CCR5 may participate in the metastatic progression of breast cancer [
12]. In the current studies, we show that the humanized monoclonal antibody leronlimab efficiently blocks ligand-induced Ca
2+ signaling, cellular invasion, and tumor metastasis. Prior findings had shown that CCR5 small-molecule antagonists (maraviroc and vicriviroc) block metastasis of human breast cancer xenografts (MDA-MB-231 cells) [
12,
13]. The current studies extend these findings by demonstrating the humanized monoclonal antibody to CCR5, leronlimab, efficiently bound CCR5 expressed on human breast cancer cells, blocked ligand-induced Ca
2+ signaling, and inhibited Matrigel invasion of breast cancer cells. Furthermore, leronlimab reduced tumor metastasis in immune-deficient mice. In a subset of mice with established TNBC lung metastasis, leronlimab reduced the metastatic tumor burden and increased overall survival. As leronlimab has been well tolerated in the HIV patient population without significant drug-related adverse events [
18], the current studies suggest leronlimab may have clinical application.
The current studies showed that leronlimab blocks CCR5 signaling, assessed by calcium release in response to several distinct ligands. Upon binding of the ligand, the conformational change in CCR5 dissociates the Gαi and the Gβγ subunits, inducing downstream signaling. The activation of Ca
2+ signaling and cellular migration by CCR5 is preserved in both immune cells [
23] and cancer cells [
12,
13]. CCR5 binds many ligands which are overexpressed in the tumor microenvironment including CXCL13 (BCA-1), CCL3 (MIP1α), CCL3L1, CCL4 (MP-1β), CCL8 (MCP2), CCL11 (eotaxin), CCL13 (MCP-4), and CCL16 (HCC-4). Elevated levels of CCL5 indicate poor prognosis in breast cancer [
33,
34], pancreatic cancer [
35], cervical cancer [
34], prostate cancer, ovarian cancer [
36], and gastric cancer [
14,
37].
Herein, leronlimab enhanced cell killing by the DNA damaging agent doxorubicin. It is likely that two previously described mechanisms contribute to these findings. Firstly, CCR5 induces both homologous and non-homologous DNA repair [
13]. Secondly, cell survival signaling pathways, ribosomal biogenesis, and PI3K/Akt are induced by CCR5 when analyzed by single-cell analysis of breast cancer cells. Many transcripts were induced (> 1000-fold) by the expression of CCR5 when compared to neighboring CCR5
− breast cancer cells [
13]. The induction of the PI3K pathway and thereby PDK1 and serine/threonine kinase protein kinase B (AKT) pathway, by CCR5, in turn, induces cell survival, glycolysis, cell proliferation, growth and proliferation of progenitor and stem cells, immune cell differentiation, and the release of eIF4E to promote cap-dependent translation [
12,
13].
A substantial number of studies have provided evidence in other systems that CCR5 participates in the important anti-tumor immune response. In the current studies, leronlimab restrained the development of tumor metastasis in murine xenografts in Nu/Nu mice which lack functional T cells [
38]. The nude mouse (nu or Hfh11nu or Foxn1nu) lack a thymus due to a mutation in the
FOXN1 gene. The absence of a thymus means that there is no production of T cells; therefore, they are unable to activate the different types of immune responses (adaptive) during the implantation of cancer cells. These mice lack antibody formation, cell-mediated immune responses, and delayed-type hypersensitivity responses but produce NK cells [
39], resulting in a reduced capability of killing virus-infected or malignant cells [
38]. Our studies suggest therefore that T cell participation is not necessary for the anti-tumor function of leronlimab observed in the current studies but do not exclude a potential role for NK cells which express CCR5 [
18]. Furthermore, as leronlimab is a humanized antibody that does not bind murine cells, it is most likely the effect seen with leronlimab is mediated directly on the human breast cancer cells, rather the local murine tumor environment. That said, evidence supports a model in which additional immune functions are regulated by CCR5 and T cells in other settings. CCL5 recruits CCR5-expressing TAMs [
40,
41]. T cells participate in the anti-tumor immune responses, in part through CCR5-dependent regulation of macrophage differentiation [
42]. The recruitment of immune cells, including tumor-infiltrating lymphocytes (TILs), MDSCs, tumor-associated macrophages (TAMs), innate lymphoid cells (ILCs), Tregs [
43], mesenchymal stem cells (MSCs), and immature dendritic cells (DCs), contributes to tumor-induced immunosuppression [
44]. Many of these cell types express CCR5 and/or produce ligands for CCR5 [
18]. Prior studies showed the small molecule CCR5 inhibitor maraviroc reduced MDSC-induced colon cancer metastasis [
45]. In the phase 1 pilot MARACON study, patients with advanced-stage metastatic colorectal cancer that were refractory to current therapies [
46] were treated with maraviroc. CCR5 inhibition correlated with reduced proliferation and an anti-tumoral macrophage polarized M1 morphology [
46], although more complex interactions occur with PD-1- and CTLA-4-positive cells surrounding tumors with patchy CCR5 expression [
47].
The current studies extend prior studies demonstrating the importance of CCR5 in breast tumor metastasis prevention and by showing for the first time a reduction in the volume of established metastasis with life extension. The requirement for CCR5 in oncogene-induced cellular proliferation was supported by transgenic studies in which MMTV-PyMT-induced mammary tumors were reduced in
CCR5−/− mice [
48]. Multiple CCR5-mediated pathways may contribute to tumor progression including MDSC [
49], vascularity, and lymphangiogenesis [
50,
51]. CCR5 siRNA did not reduce the metastatic phenotype of MDA-MB-231 cells in the absence of additional MDSC [
52], endothelial cells produce CCL5, and augmented breast cancer metastasis in another study [
49]. In addition, CCR5 inhibitors also reduced lymphangiogenesis in triple-negative breast cancer (TNBC) cell line xenografts [
50,
51]. Other approaches to restrain tumor metastasis via CCR5 inhibition include targeting CCL5 in the bone marrow via nanoparticle-delivered expression silencing, in combination with maraviroc, which augmented anti-tumor immunity [
53].
It is likely that CCR5 plays a broader role in governing cancer metastasis as maraviroc and vicriviroc reduced prostate cancer cell metastasis to the bones, brain, and viscera in immune-competent mice [
22] and reduced metastasis or cellular migration in glioblastoma [
54] and a variety of other malignancies [
18]. Prior studies had shown that CCR5 induces cancer cell homing to metastatic sites [
12,
55], augments the pro-inflammatory pro-metastatic immune phenotype [
46], and enhances DNA repair [
13], providing aberrant cell survival and resistance to DNA-damaging agents. The current studies, showing a reduction in the volume of established breast cancer metastasis with life extension, provide support for a controlled clinical intervention study using leronlimab in patients with TNBC.
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