Development of distant metastases involves a complex multistep biological process termed the
invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs [
41]. This process is inefficient because it has been estimated that less than 1% of cancer cells will be successful in establishing clinically detectable metastatic lesions [
42]. Specifically in breast cancer, BT-MICs must go through EMT, invade the extracellular matrix, intravasate and survive in the systemic circulation, extravasate at the metastatic site, and finally seed in the new microenvironment [
42‐
44]. Importantly, each of these events is driven by the accumulation of genetic and/or epigenetic alterations within cancer cells necessary for the clonal selection and expansion of BT-MICs that ultimately give rise to distant metastases [
44]. Several lines of evidence support the hypothesis that BT-MICs might be found within subpopulations of BTICs [
46]. In support of this hypothesis, it has been shown that oncogenic pathways such as MAPK, AURKA, and NOTCH that induce EMT and expansion of BTICs also promote onset of distant metastases [
5,
29,
47,
48]. The characterization of the precise role of each of these oncogenic pathways in the sequential stepwise events that typify the invasion-metastasis cascade will be essential for the development of precise therapeutic strategies aimed at eradicating distant metastases.
In this study, we uncovered the linkage between NOTCH3 expression and development of distant metastases in experimental breast cancer models. First, we used luminal ER
+ MCF-7 and variant cells with constitutively active Raf-1/MAPK signaling (vMCF-7
∆Raf1) to establish in vivo the association between aberrant Raf-1/MAPK signaling, CIN, and onset of distant metastases. In agreement with our previous studies [
5,
29], only vMCF-7
∆Raf1 tumor xenografts developed spontaneous lung metastases, corroborating the causal role of aberrant activation of Raf-1/MAPK pathway in promoting metastatic lesions. Significantly, ex vivo cancer cells isolated from lung metastases (vMCF-7
∆Raf1 1GX-M) showed a normal centrosome phenotype and clonal chromosomal aberrations compared with matching vMCF-7
∆Raf1 1GX parental cells that exhibited centrosome amplification and nonclonal chromosomal aberrations resulting in CIN. These findings demonstrate in vivo that loss of centrosome amplification is linked to restoration of chromosomal stability resulting in the clonal expansion of metastatic cancer cells. Moreover, they support the genomic convergence model proposed for tumor progression in which CIN initially imposed during tumorigenesis becomes suppressed when cancer cells have acquired the suitable chromosome compositions and gene dosage that will lead to the successful establishment of distant metastases [
48]. Because it has been hypothesized that BT-MICs are late stages BTIC subclones with higher stemness capacity [
45], we cultured vMCF-7
∆Raf1 1GX-M and matching vMCF-7
∆Raf1 and vMCF-7
∆Raf1 1GX parental cells under nonadherent conditions to form MPS as an in vitro surrogate assay of self-renewal capacity. vMCF-7
∆Raf1 1GX-M cells showed the highest efficiency in MPS formation compared with matching parental cells. This increased self-renewal capacity was linked to loss of the CD24 epithelial marker in vMCF-7
∆Raf1 1GX-M MPS. These results demonstrate that chromosomal stable vMCF-7
∆Raf1 1GX-M cells have acquired higher self-renewal and CD24
−/low basal-like plasticity that plays a critical role in EMT, cancer cell seeding, and metastatic growth to secondary organs [
29]. Next, we wanted to establish whether chromosomal stability and high self-renewal capacity of vMCF-7
∆Raf1 1GX-M cells was linked to an exclusive metastatic signature. To answer this question, we performed unbiased comparative transcriptomic and functional gene enrichment analyses between MPS vMCF-7
∆Raf1 1GX-M (that show CD24
−/low) and highly invasive CD24
−/low basal-like cells isolated from matching vMCF-7
∆Raf1 tumor xenografts as previously demonstrated [
29]. Functional gene enrichment analysis identified a noncanonical NOTCH3 reprogramming network that was upregulated in vMCF-7
∆Raf1 1GX-M MPS. The NOTCH3 network comprised nine genes encoding for transcriptional factors with high oncogenic activity (HES1, FOSB, JUN, EGR1, EGR3, MYC, TFDP2, ATF3, PGR). This reprogramming network included the NOTCH downstream target HES1, suggesting that NOTCH3/HES1 stemness signaling may play a central role in promoting the survival and seeding of BT-MICs to secondary organs. Because detection of cancer cell seeding to secondary organs and onset of micrometastases is clinically challenging, expression of the NOTCH3 metastatic signature in circulating tumor cells may have promising clinical relevance in predicting early onset of distant metastases in patients with breast cancer. Importantly, the majority of vMCF-7
∆Raf1 1GX-M cells were strongly positive for NOTCH3 staining by immunofluorescence assay, demonstrating that NOTCH3 expression was restricted to clonal metastatic breast cancer cells. vMCF-7
∆Raf1 1GX xenografts exhibiting CIN showed tumor cell heterogeneity for NOTCH3 expression, indicating that NOTCH3
high-expressing subclones may arise from the primary tumor and promote distant metastasis owing to their higher stemness capacity, in agreement with the recent finding that metastatic clones disseminate early from primary breast tumors [
49]. On the basis of these results, we developed unique NOTCH3-knockout breast cancer cells (vMCF-7
Raf-1 1GX
CRISPR-NOTCH3) to abrogate NOTCH3 expression and evaluate the causative role of NOTCH3 signaling in promoting stemness and invasive properties of vMCF-7
∆Raf1 1GX cells. NOTCH3 expression was required to induce in vitro self-renewal capacity and a CD44
high/CD24
low breast cancer stemlike phenotype in vMCF-7
∆Raf1 1GX cells. Because we and others have demonstrated that CD44
high/CD24
low BTICs also show an ERα
low/− basal-like phenotype [
29,
37,
38], we assessed ERα in MPS derived from vMCF-7
Raf-1 1GX
CRISPR-NOTCH3 and parental cells. Whereas MPS derived from vMCF-7
Raf-1 1GX cells lacked ERα expression, MPS resulting from vMCF-7
Raf-1 1GX
CRISPR-NOTCH3 cells exhibited restoration of ERα expression, compatible with previous studies that demonstrated the role of NOTCH3 signaling in suppressing ERα expression [
38]. Because high ALDH1 activity has been linked to stemness, early onset of distant metastases, and poor prognosis in breast cancer [
39], we performed an ALDEOFLUOR assay that accurately identifies highly tumorigenic cancer cells with elevated ALDH1 activity. Importantly, vMCF-7
Raf-1 1GX
CRISPR-NOTCH3 cells showed minimal ALDH1 activity compared with parental vMCF-7
Raf-1 1GX cells, supporting the role of NOTCH stemness signaling in inducing ALDH1 activity and an increased metastatic behavior [
50]. Next, we defined whether inhibition of self-renewal capacity and tumor stemness was functionally linked to loss of invasive capacity. An in vitro real-time invasion assay showed that abrogation of NOTCH3 expression significantly inhibited the invasive capacity of vMCF-7
Raf-1 1GX cells, indicating that NOTCH3 signaling pathway is necessary to promote a more aggressive phenotype. Because we have previously demonstrated the causative role of aberrant AURKA activity in driving the development of breast cancer metastases [
29], we aimed to establish whether NOTCH3 expression was required to mediate AURKA-induced highly invasive capacity of vMCF-7
∆Raf1 1GX cells. Forced expression of AURKA in vMCF-7
∆Raf1 1GX cells increased NOTCH3 expression and their in vitro invasive capacity. Conversely, AURKA overexpression in vMCF-7
Raf-1 1GX
CRISPR-NOTCH3 cells failed to restore a highly invasive phenotype, demonstrating that NOTCH3 expression is required to mediate AURKA-induced high metastatic potential. Moreover, these results highlight a novel mechanistic linkage between AURKA and NOTCH3 oncogenic pathways that is critical to development of a fully metastatic phenotype in breast cancer cells. To define in a different breast cancer model whether increased expression of NOTCH3 was restricted to metastatic cancer cells, we employed MDA-MB-231 TNBC cells that exhibit a CD44
high/CD24
low basal-like phenotype and elevated endogenous MAPK activity [
34,
52]. Significantly, the percentage of ex vivo MDA-MB-231 cells isolated from lung metastases (MDA-MB-231 LM) expressing NOTCH3 was higher than matching MDA-MB-231 parental cells, suggesting that NOTCH3 signaling is also required for the metastatic seeding and growth of TNBC cells. These results are in agreement with a recent study that demonstrated the role of the NOTCH3 signaling pathway in promoting the growth of basal-like breast cancer cells [
51‐
53]. Complementary to our vMCF-7
Raf-1 model, targeting of NOTCH3 impaired the in vitro invasive capacity of MDA-MB-231 LM cells, demonstrating the key role of NOTCH3 expression in promoting the TNBC highly invasive phenotype. Next, we aimed to determine whether inhibition of NOTCH signaling decreased the metastatic capacity of MDA-MB-231 LM cells. In vitro treatment of MDA-MB-231 LM cells with the pan-NOTCH inhibitor LY-411575 resulted in the inhibition of cancer cell seeding and onset of experimental lung metastases, demonstrating that NOTCH pharmacologic targeting interferes with late stages of the invasion-metastasis cascade in NOTCH3-expressing breast cancer cells. Importantly, MDA-MB-231 LM and matching parental cells expressed nominal levels of NOTCH1 and NOTCH2, suggesting that LY411575-mediated inhibition of cancer cell seeding and metastatic growth was primarily linked to inhibition of the NOTCH3 signaling pathway.
Owing to the limited translatability of established cancer cells, and to corroborate the central role of NOTCH3 in driving a metastatic phenotype in clinically relevant models, we established unique TNBC cells (TNBC-M25) isolated from a patient-derived brain metastasis xenograft model. TNBC-M25 cells showed high expression of phospho-AURKA and NOTCH3, whereas NOTCH1 and NOTCH2 levels were low, suggesting that the AURKA/NOTCH3 oncogenic axis plays a major role in promoting their metastatic phenotype. To test this hypothesis, we reduced NOTCH3 expression by lenti-shRNAs in TNBC-M25 cells. Reduction of NOTCH3 expression impaired self-renewal capacity, resulting in a significant shrinkage of TNBC-M25 MPS, confirming the essential role of the NOTCH3 signaling pathway in promoting tumor stemness. Moreover, inhibition of NOTCH3 expression also reduced in vitro the invasiveness of TNBC-M25 cells. These results validated our findings in vMCF-7∆Raf1 1GX cells that the NOTCH3 signaling pathway is downstream of AURKA and is required to promote breast cancer cells’ aggressiveness.