COX-2 is overexpressed in 40% of cases of invasive breast carcinoma and has been implicated in multiple steps during breast tumor progression, including primary tumor growth, metastasis, angiogenesis, and immune evasion. Even though COX-2 inhibitors have been proven to attenuate breast tumor growth and metastasis in preclinical models, the clinical benefit of COX-2 inhibitors in breast cancer patients remain elusive. Meta-analyses of aspirin use showed a 9–30% reduced risk of breast cancer incidence [
34]. Regular use of COX-2 inhibitors was also associated with 60–70% reduced risk of breast cancer for women at familial or genetic risk [
35]. Celecoxib (Celebrex) has been approved by FDA to treat arthritis patients, and its potential to treat cancer patients such as breast cancer is still under investigation. A clinical study in breast cancer showed that pre-operative celecoxib treatment sets up transcriptional programs supporting anti-tumor activity [
36]. Other trials demonstrated that combination of celecoxib with aromatase inhibitors in the neoadjuvant treatment is effective in reducing breast tumor size and area [
23,
24]. Results from a randomized phase II trial of celecoxib plus exemestane compared with exemestane alone in patients with hormone-sensitive breast cancer (
n = 111) suggested a trend in favor of combination therapy, evidenced by an approximately twofold longer duration of clinical benefit in patients receiving the combination treatment [
37]. However, a phase III multicenter double-blind randomized trial of celecoxib versus placebo in primary breast cancer patients showed no benefit of celecoxib in BC patients [
25]. Several possibilities might explain the mixed results produced by these studies. First, there were no proper stratification criteria established for breast cancer patients receiving COX-2 inhibitor treatment. Also, little is known regarding mechanisms for underlying COX-2 inhibitor insensitivity/resistance in breast cancer. Thus, the aim of our study is to characterize the role of COX-2 and COX-2-associated genes in regulating breast cancer tumorigenesis as well as to identify COX-2 inhibitor resistance genes. We mainly focused on an extremely aggressive subtype of breast cancer, TNBC in this study, since patients with TNBC exhibit poor prognosis and lack of specific actionable molecular targets. Leveraging existing large breast cancer databases and cohorts with genomic and transcriptomic profiles as well as clinical data, we were able to develop a systematic data mining strategy to identify COX-2-associated genes in TNBC that are correlated with its aggressive features and breast cancer resistance to COX-2 inhibitor. Using CRISPR/Cas9 gene editing tools and preclinical models of breast cancer, we functionally validated the identified genes and addressed their roles and contributions to (1) breast cancer metastasis and (2) breast tumor resistance to COX-2 selective inhibitor, celecoxib. We found 10 genes (
TPM4,
RGS2,
LAMC2,
SERPINB5,
KLK7,
MFGE8,
KLK5,
ID4,
RBP1,
SLC2A1) that regulate TNBC distant lung metastasis in vivo, among which 6 genes (
TPM4,
RGS2,
SERPINB5,
MFGE8,
KLK5,
ID4) individual KO led to more than 90% reduction of lung metastatic area. We also showed that individual knockouts of
MFGE8,
KLK5, and
KLK7 resulted in increased sensitivity to celecoxib in TNBC both in vitro and in vivo. These results demonstrate the robustness and power of our multi-level in silico data analysis strategy combined with in vitro
/in vivo functional validation. This systematic approach could be applied to other studies with the goal of investigating any gene/pathway-associated gene network and their regulation in any specific step of tumorigenesis as well as mechanisms of acquired drug resistance. We found that
MFGE8 gene KO led to more than 90% reduction of TNBC lung metastatic area in our preclinical model.
MFGE8 gene encodes for the milk fat globule-EGF factor 8, a secreted glycoprotein that mediates adhesion to integrin-expressing cells [
38].
MFGE8 has been shown to regulate tumorigenesis through multiple mechanisms: enhancing phagocytosis of apoptotic cells by endothelial and epithelial cells [
39,
40]; inducing tumor mesenchymal phenotype through the activation of Akt [
41]; as well as promoting vascular endothelial growth factor (VEGF)-induced angiogenesis by binding to avb3/b5 integrins [
42]. Since no changes of cell phenotype and proliferation rate were observed upon
MFGE8 deletion in TNBC cell lines, we hypothesize that impaired
MFGE8-mediated angiogenesis might be the mechanism underlying reduced lung metastasis in
MFGE8 KO tumors. Interestingly, COX2/PGE2 pathway was found to regulate tumor angiogenesis in a VEGF-independent manner and mediate refractoriness to VEGF/VEGFR2 inhibition [
43]. Similar observations were made by another research group, showing that COX-2 inhibition improves the efficacy of antiangiogenic therapy in breast cancer and colorectal cancer preclinical models [
44]. Since MFGE8 was not upregulated in celecoxib-resistant TNBC cells, the potential synergistic effects of MFGE8 deletion with COX-2 inhibition on suppressing TNBC primary tumor growth might be due to convergent anti-angiogenesis pathway rather than overcoming celecoxib resistance and should be investigated in future studies. Our results also suggest that combination treatments aiming at disabling both COX-2 and MFGE8 could represent a therapeutic strategy for the treatment of TNBC. Although MFGE8 has been shown to be overexpressed in TNBC compared with non-TNBC patients [
45], it is also an essential gene for the breast involution process [
46]. Thus, precise examination and attentive care will be required when targeting MFGE8 in clinical settings to avoid any potential side effects related to abnormal mammary gland remodeling.
Kallikrein-related peptidase 5 and 7 (KLK5 and KLK7) are members of a subgroup of 15 homologous secreted serine proteases and are highly expressed in endocrine or hormone-responsive tissues including breast, ovary, and skin [
47,
48]. KLK5 has been shown to activate KLK7 in vitro and is considered as the physiological activator of KLK7 [
49]. Several studies have shown that KLK5 and KLK7 serve as serological biomarkers and indicators of poor prognosis in breast and ovarian cancer [
50‐
54]. Consistent with these findings, we found high expression of KLK5 and KLK7 to correlate with aggressive pathological features and poor patient outcomes in TNBC. In addition, kallikrein-regulated extracellular proteolysis is implicated in many cancer-related processes, such as tumor cell growth, invasion, metastasis, and angiogenesis [
55]. Indeed, KLK5 has recently been shown to cleave ECM (collagens type I, II, III, IV, fibronectin, and laminin) and adhesion molecules (fibrinogen and vitronectin), suggesting a role in tumor invasion and angiogenesis [
47]. In our study, both
KLK5 and
KLK7 gene KOs in TNBC cell lines block distant lung metastasis in vivo, demonstrating their pro-tumorigenic function. Moreover, as a COX-2 associated gene in TNBC, we found
KLK5 and
KLK7 gene KOs to restore tumor cell sensitivity to celecoxib both in vitro and in vivo. Although there is no literature showing the direct interactions between KLK and COX-2 signaling pathway, the cooperativity between COX-2 inhibition and KLK KO in reducing tumor growth is worth further investigation. A better understanding of the crosstalk between the COX-2 pathway and KLK pathways will be useful for future design and personalization of novel COX-2 inhibitor-based combination therapies in clinical settings. The potential of KLK5 and KLK7 as therapeutic targets in cancer has led to advances in the development of the first generation of KLK-based inhibitors. As of current, these pharmacological efforts are mainly directed toward the design of small-molecule inhibitors, such as triazole derivatives [
56] and other compounds identified in high-throughput screening of large chemical libraries as well as peptide/protein-based inhibitors [
57‐
59]. Thus, it will be interesting to further test the use of the various KLK inhibitors in TNBC, using combi-therapy with the FDA-approved anti-COX-2 drugs.