Introduction
Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. IBC patients often develop metastasis in brain, bones and soft tissue with variable frequency, being the most aggressive features described in triple-negative (TN) IBC. Although IBC accounts for 1–6 % of all breast cancer cases in the USA and up to 20 % of all breast cancers globally [
1], its incidence is dramatically increasing [
2,
3]. Due to its propensity to rapidly metastasize, IBC is responsible for a disproportionate number of breast cancer-related deaths [
1]. Approximately, 30 % of IBC patients have distant metastases at the time of diagnosis, in contrast to 5 % of patients with non-IBC breast cancers. IBC occurs commonly in patients under the age of 50 years and since it does not present as a lump is often misdiagnosed as an infection [
4]. The clinical symptoms of IBC are very distinct from other types of breast cancer and involve the rapid onset of changes in the skin overlying the breast, including edema, redness and swelling, exhibiting a wrinkled and orange-peel appearance of the skin defined as peau d’orange [
5]. This peculiar presentation that mimics an inflammation is associated with the invasion of aggregates of tumor cells, defined as tumor emboli into the dermal lymphatics, causing an obstruction of the lymph channels [
6]. IBC tumor emboli are non-adherent cell clusters that rapidly spread by a continuous passive dissemination [
7], thus favoring both distal metastasis and local recurrence. Although IBC, like non-IBC breast cancers, is a heterogeneous disease and can occur as any of the five molecular subtypes, they are most commonly either ErbB2 overexpressing or triple negative [
8].
Few models are currently available to evaluate the peculiar biology of IBC and improve our understanding of the factors associated with early activation of the metastatic process in this disease. The majority of the IBC studies have been performed using the cell lines SUM149 and SUM190 [
9]. SUM149 cells are triple negative and SUM190 cells are ER/PR negative and ErbB2 positive [
10‐
13]. Other less studied IBC cell lines are MDA-IBC3, KPL4 and WIBC-9—all ER/PR-negative, ErbB2-positive—and the triple-negative Mary-X xenografts [
14‐
18]. In this work, we report the isolation and characterization of a new human triple-negative IBC model, FC-IBC02. Genomic and expression studies were performed in FC-IBC02 and other IBC cell lines to understand the metastatic process of IBC and determine putative targets for therapy.
Discussion
We have developed a new IBC cell line, FC-IBC02, derived from the pleural effusion of a woman with rapidly progressing secondary IBC. These tumor cells and xenografts in mice showed the same markers as the original pleural effusion in the patient from whom they were isolated. FC-IBC02 breast tumor xenografts grew rapidly and all mice developed spontaneous metastases within lungs and lymph nodes. FC-IBC02 cells were able to recapitulate the peculiar metastatic process observed in IBC patients, as shown by the formation of tumor emboli within lymphatics of SCID mice. Remarkably, injection of FC-IBC02 tumor cells via either the intraperitoneal or intracardiac routes resulted in the formation of brain metastases.
The symptoms of IBC are caused by the invasion of aggregates of tumor cells (tumor emboli) into the dermal lymphatics, causing an obstruction of the lymph channels [
6]. Although, tumor emboli are also found in non-IBC tumors, they are more frequent and high in number in IBC [
25]. Tumor emboli expressed cell–cell adhesion molecules that maintain the tumor cells together. Approximately, 90 % of human IBCs are associated with increased E-cadherin indicating that the gain of E-cadherin axis contributes to the IBC phenotype [
18]. FC-IBC02 cells, breast tumor xenografts and metastatic lesions within lungs in SCID mice showed strong expression of E-cadherin and β-catenin in cell membranes. For most non-IBC tumors, loss of E-cadherin and acquisition of mesenchymal phenotype have been associated with increased invasion and metastatic potential, although in IBC tumor cells gained a survival benefit from the expression of E-cadherin mediating the formation of the characteristic tumor emboli. Moreover, FC-IBC02 cells showed strong expression of the membrane tetraspanin 24 (TSPAN24/CD151). Collectively, these results demonstrate that the FC-IBC02 tumor emboli express E-cadherin, β-catenin and TSPAN24/CD151 suggest that these adhesion molecules may have a functional role in maintaining the tight aggregation of cells within the tumor emboli. The maintenance of cell–cell adhesions allow the migration of tumor cells through the lymphatic and blood vessels as clusters, thus supporting the hypothesis that cohesive or collective migration may provide a survival advantage by protecting cells from immune attack or shear forces during transit through the circulation [
26]. The presence of clusters of circulating tumor cells (CTCs) have been observed in the blood of IBC patients (data not shown).
The expression of epithelial markers by IBC cells is paradoxical to the current hypothesis that metastasis occurs as part of the process of epithelial–mesenchymal transition (EMT). Through the EMT process, epithelial cells lose cell–cell contact and cell polarity, downregulate epithelial-associated genes and acquire a mesenchymal phenotype; this cellular process culminates with single cells having increased motility, invasiveness and metastatic capacity. FC-IBC02 cells showed some, but not all the characteristic expression patterns of EMT; these cells expressed vimentin (
VIM) and the EMT-transcription factors SNAI2 and TWIST1, although they showed strong expression of the epithelial markers EpCAM, MUC1 and E-cadherin. It has been suggested that SNAI2 and TWIST1 are probably responsible for maintaining the stem cell state rather than inducing mesenchymal marker expression [
27,
28]. Recent studies suggest that EMT is not a pre-requisite for tumor cell invasion and cells can move in a collective manner dependent on maintenance of cell–cell adhesion molecules [
29].
At the genomic level, FC-IBC02 cells and other IBC cells showed extensive chromosomal copy number changes indicating a higher degree of genomic instability in IBC that is in agreement with their high grade. NOTCH3 was amplified in FC-IBC02, and several genes from the Notch signalling pathway were upregulated in this cell line. Recently, it was shown that Notch 3 was also activated in Mary X [
30]. Furthermore, IBC cells showed amplification of the 8q chromosomal arm where the oncogene
MYC and ATAD2, a cofactor for MYC, are located.
MYC showed 2.5–7 copies in IBC cells.
MYC amplification is one of the most consistent markers of adverse prognosis for cancer and is more often amplified in IBC than non-IBC tumors [
31]. MYC expression was higher in the triple-negative IBC cell lines FC-IBC02 and SUM149 when compared with the triple-negative non-IBC MDA-MB-231 and MDA-MB468, and the ErbB2 + IBC cells SUM190 and MDA-IBC3. MYC has a large number of targeted genes and it upregulates genes involved in cell growth and proliferation [
32]. ATAD2 was amplified and upregulated in FC-IBC02; it was shown that this gene is usually overexpressed and amplified in aggressive tumors [
33]. ATAD2 associates through its bromodomain (BRD) with histone H3 acetylated at Lys 14 during late mitosis, regulating the expression of genes required for cell cycle progression [
33]; recently, specific inhibitors to BRD-containing proteins have been developed [
34‐
36]. Targeting ATAD2 through the use of specific inhibitors to its BRD domain will be useful as therapeutic targets in MYC-driven tumors.
Metadherin (MTDH), a gene related to anoikis resistance, located at 8q, was also amplified and upregulated in FC-IBC02 cells. Anchorage-independent survival or anoikis resistance is characteristic of tumor cells and contributes to metastasis [
37]. All tumor cells that eventually form metastases at distant sites must survive in circulation and then in metastatic organs, and all of these microenvironments are quite distinct from that of the breast. The ability of tumor cells to survive in these altered matrix conditions is known as anoikis resistance (adhesion-independent growth) and is in contrast to normal epithelial cells, which undergo cell death (anoikis) when deprived of signals from the normal extracellular matrix [
37]. MTDH functions as a downstream mediator of the transforming activity of oncogenic Ha-Ras and c-MYC and promotes lung, bone and brain metastasis [
38]. Interestingly, another gene previously reported to contribute to metastasis via regulation of anoikis resistance is the focal adhesion kinase FAK1 (or PTK2) [
37], which shows 2.5–7 copies in IBC. FAK1 is a non-receptor tyrosine kinase that localizes to focal adhesions and controls a number of cell pathways including proliferation, viability and survival. Its overexpression was linked to anoikis resistance [
39]. FAK1 mRNA and protein levels are absent or low in normal tissue and benign neoplasms, and are upregulated in invasive and metastatic tumors [
40]. Elevated FAK1 levels have been reported in many tumors such as epithelial tumor of the breast [
39]. We have studied FAK1 in the triple-negative IBC cell lines FC-IBC02 and SUM149, and ErbB2-positive cell lines SUM190 and KMO-015, and this protein showed to be activated in these cells (phosphorylated in Y397). Studies for evaluating a new dual ALK-FAK1 inhibitor in IBC cells and xenograft models are ongoing in our laboratory.
In summary, the gene signature and phenotypic characteristics of FC-IBC02 and other IBC cell lines suggest that IBC exhibits characteristics of epithelial plasticity, where tumor cells retain an epithelial phenotype through E-cadherin and EpCAM expressions, while simultaneously expressing markers of cancer stem cells. FC-IBC02 xenografts represent an ideal model for studying spontaneous metastases in lungs and lymph nodes and brain metastases by the intracardiac route. Moreover, FC-IBC02 and other IBC cells demonstrated amplification of MYC, ATAD2, CD44, NOTCH3, ALK and FAK1 indicating that these genes could be used as potential target therapies against IBC.