Inhibition of Cdk2 kinase activity selectively targets the CD44+/CD24−/Low stem-like subpopulation and restores chemosensitivity of SUM149PT triple-negative breast cancer cells

Inflammatory breast cancer (IBC) is an angioinvasive and most aggressive type of advanced breast cancer characterized by rapid proliferation, chemoresistance, early metastatic development and poor prognosis. IBC tumors display a triple-negative breast cancer (TNBC) phenotype characterized by centrosome amplification, high grade of chromosomal instability (CIN) and low levels of expression of estrogen receptor α (ERα), progesterone receptor (PR) and HER-2 tyrosine kinase receptor. Since the TNBC cells lack these receptors necessary to promote tumor growth, common treatments such as endocrine therapy and molecular targeting of HER-2 receptor are ineffective for this subtype of breast cancer. To date, not a single targeted therapy has been approved for non-inflammatory and inflammatory TNBC tumors and combination of conventional cytotoxic chemotherapeutic agents remains the standard therapy. IBC tumors generally display activation of epithelial to mesenchymal transition (EMT) that is functionally linked to a CD44+/CD24−/Low stem-like phenotype. Development of EMT and consequent activation of stemness programming is responsible for invasion, tumor self-renewal and drug resistance leading to breast cancer progression, distant metastases and poor prognosis. In this study, we employed the luminal ER+ MCF-7 and the IBC SUM149PT breast cancer cell lines to establish the extent to which high grade of CIN and chemoresistance were mechanistically linked to the enrichment of CD44+/CD24low/− CSCs. Here, we demonstrate that SUM149PT cells displayed higher CIN than MCF-7 cells characterized by higher percentage of structural and numerical chromosomal aberrations. Moreover, centrosome amplification, cyclin E overexpression and phosphorylation of retinoblastoma (Rb) were restricted to the stem-like CD44+/CD24−/Low subpopulation isolated from SUM149PT cells. Significantly, CD44+/CD24−/Low CSCs displayed resistance to conventional chemotherapy but higher sensitivity to SU9516, a specific cyclin-dependent kinase 2 (Cdk2) inhibitor, demonstrating that aberrant activation of cyclin E/Cdk2 oncogenic signaling is essential for the maintenance and expansion of CD44+/CD24−/Low CSC subpopulation in IBC. In conclusion, our findings propose a novel therapeutic approach to restore chemosensitivity and delay recurrence of IBC tumors based on the combination of conventional chemotherapy with small molecule inhibitors of the Cdk2 cell cycle kinase.


Introduction
Inflammatory breast cancer (IBC) is an angioinvasive form of breast cancer in which cancer cells block the lymphatic vessels in the skin covering the breast and it is associated with a high incidence of early nodal and systemic metastases (1). This type of breast cancer is called 'inflammatory' because the breast often looks swollen and red, resembling an inflammatory condition (2). Although IBC accounts for only 2-5% of all breast cancers, IBC tumors show a poor prognosis with 40% five-years survival rate versus 87% for all breast cancers combined, making IBC a priority area for the development of new therapeutic strategies (3). IBC tumors display a triple negative breast cancer (TNBC) phenotype characterized by high chromosomal instability (CIN) and low levels of expression of estrogen receptor α (ERα), progesterone receptor (PR) and HER-2 tyrosine kinase receptor (4). Since the TNBC cells lack these receptors necessary to promote tumor growth, common treatments such as endocrine therapy and molecular targeting of HER-2 receptor are ineffective for this subtype of breast cancer (5). Employing conventional chemotherapy to treat TNBC tumors is still an effective option because TNBC tumors may respond even better to chemotherapeutic agents in the earlier stages than many other forms of breast cancer (6). Unfortunately, TNBC tumors will likely develop chemoresistance leading to tumor progression, metastatic spreading to distant organs and poor outcome (7).
Germline mutations in the BRCA1 and BRCA2 breast cancer susceptibility genes have been associated with up to 15% of TNBC, and TNBC accounts for 70% of breast tumors arising in BRCA1 mutation carriers and 16-23% of breast tumors in BRCA2 carriers (8). Because BRCA1 and BRCA2 tumor suppressor genes play a key role in the control of genomic stability through regulation of DNA repair and centrosome duplication (9)(10)(11), these findings explain the causal role of BRCA1 and BRCA2 mutations in the development of high CIN commonly observed in TNBC tumors. Moreover, TNBC tumors frequently overexpress cyclin E, a late G1 cyclin that binds and activates cyclin-dependent kinase 2 (Cdk2) leading to phosphorylation and inactivation of retinoblastoma (Rb) essential for the G1-S phase transition of the cell cycle (12,13). Importantly, aberrant activation of cyclin E/Cdk2 complex plays a key role in the development of centrosome amplification, CIN and breast cancer progression and may represent an attractive 'druggable oncogenic signaling' for the treatment of TNBC tumors lacking molecular targeted therapy (14)(15)(16). Recent studies have demonstrated that breast carcinomas are composed of a singular subpopulation of cells harboring stem-like properties termed cancer stem cells (CSCs) (17). CSCs show activation of epithelial to mesenchymal transition (EMT) programming and acquire a basal-like CD44 + /CD24 low/phenotype with increased capacity for self-renewal, invasion, drug resistance and tumor progression (18)(19)(20). The discovery of CD44 + /CD24 low/-CSCs has generated excitement because this subpopulation of cancer cells may represent a source of therapeutic failure to anticancer drugs. Significantly, it has been demonstrated that treatment of breast tumors with conventional chemotherapeutic agents enriched the CD44 + /CD24 low/subpopulation conferring resistance to the initial treatment and leading to the development of distant metastases (21). In agreement with these findings, TNBC tumors generally display a basal-like CD44 + /CD24 low/phenotype that clarifies their resistance to conventional chemotherapy responsible for high risk of recurrence and poor prognosis (22).
In this study we employed the luminal ER + MCF-7 and the IBC SUM149PT breast cancer cells to establish the extent to which high grade of CIN and chemoresistance was mechanistically linked to the enrichment of CD44 + /CD24 low/-CSCs. We demonstrate that overexpression of cyclin E is restricted to the stem-like CD44 + /CD24 low/subpopulation and is functionally linked to phosphorylation of retinoblastoma (Rb), centrosome amplification and genomic instability. Significantly, CD44 + /CD24 low/-CSCs displayed higher sensitivity to a specific Cdk2 inhibitor than the bulk SUM149PT cells indicating that aberrant activation of cyclin E/Cdk2 oncogenic signaling is essential for the maintenance and expansion of CD44 + /CD24 low/-CSCs subpopulation in IBC. In conclusion our findings highlight a novel therapeutic approach based on the combination of conventional chemotherapy with small molecule inhibitors of the Cdk2 cell cycle kinase to treat chemoresistant IBC tumors.

Materials and methods
Human breast cancer cell lines. The human breast cancer cell line MCF-7 was obtained from ATCC (Manassas, VA, USA), normal human mammary epithelial cells HMEC and IBC SUM149PT cancer cells were kindly provided by Dr Lingle and Dr Couch's laboratories, respectively (Mayo Clinic, Rochester, MN). All cell lines were maintained in EMEM medium containing 5 mM glutamine, 1% penicillin/streptomycin, 20 microgram insulin/ml and 10% FBS at 37˚C in 5% CO 2 atmosphere.
Cytogenetic and SKY analysis. Cell harvest and metaphase slide preparation for routine cytogenetic and spectral karyotyping (SKY) analysis were performed as previously described (23)(24)(25). Hybridization, wash and detection of the human SKYPaint ® probe (Applied Spectral Imaging; Vista, CA) were performed as recommended by the manufacturer. Image acquisition and spectral analysis of metaphase cells were achieved by using the SD200 SpectraCube™ Spectral Imaging system (Applied Spectral Imaging) mounted on a Zeiss Axioplan2 microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Images were analyzed using HiSKY analysis software (Applied Spectral Imaging).
Immunoblot and immunofluorescence assays. Immunoblot and immunofluorescence studies were performed as previously described (27). Antibodies employed for the immunoblot and immunofluorescence assays were: pericentrin (Abcam), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), P~Rb and β-actin (Sigma, St. Louis, MO). Results are derived from three independent experiments.
Chemoresistance studies. For chemoresistance studies, 5x10 4 cancer cells were plated in 6-well costar plates and cultured in complete EMEM medium for 48 h. Following 48-h incubation, cancer cells were treated with 1 µM methotrexate, 0.5 µM paclitaxel alone or in combination with 1 µM SU9516 (Sigma) for additional 72 h. Cytotoxicity of conventional chemotherapy alone and in combination with SU9516 was tested by immunofluorescence employing a cleaved-PARP antibody (Cell Signaling Technology, Danvers, MA) as a marker of apoptosis. Results are derived from three independent experiments.

Results
In view of the fact that IBC tumors commonly display higher grade of CIN compared to luminal ERα + breast tumors, we employed luminal ERα + MCF-7 and IBC SUM149PT cancer cell lines to investigate their level of chromosomal abnormalities; normal mammary epithelial HMEC cells were used as control. The SUM149PT cancer cell line was isolated from an IBC tumor and carries the 2288delT mutation that is linked to loss of BRCA1 function and represents an excellent preclinical model to study the molecular mechanisms responsible for the development of chemoresistance and tumor progression in IBC tumors (23). We performed an integrative karyotypic analysis of HMEC, MCF-7 and SUM149PT cells employing the spectral karyotyping (SKY) technology and routine cytogenetic analysis (Fig. 1A). Breast cancer cell lines were harvested and metaphase spreads for cytogenetic and SKY analyses were prepared as previously described (24). Comparison of the three cell lines showed that while MCF-7 and SUM149PT cancer cells displayed a variety of chromosomal abnormalities, HMEC cells exhibited a normal diploid karyotype. Significantly, SUM149PT cells displayed a higher rate of CIN characterized by higher percentage of structural and numerical chromosomal abnormalities compared to MCF-7 cells (Fig. 1B).
To establish the extent to which the higher level of CIN observed in the SUM149PT cancer cells was functionally linked to the presence of a stem-like CD44 + /CD24 -/Low subpopulation, FACS analysis was performed on MCF-7 and SUM149PT cancer cells to analyze the percentage of cells displaying a CD44 + /CD24 -/Low phenotype. While MCF-7 cells showed mainly a luminal CD44 -/CD24 + phenotype, 18% of SUM149PT cells exhibited a stem-like CD44 + /CD24 -/Low phenotype ( Fig. 2A). Because CIN in breast cancer is mechanistically linked to development of centrosome abnormalities, we analyzed the grade of centrosome amplification in bulk SUM149PT cancer cells versus the stem-like CD44 + /CD24 -/Low subpopulation isolated by FACS sorting assay. Centrosome amplification in cancer cells was examined by labeling the centrosome size with pericentrin, an oncoprotein that is localized in the pericentriolar material (25). Significantly, the CD44 + /CD24 -/Low subpopulation revealed higher centrosome amplification compared to bulk SUM149PT cancer cells ( Fig. 2B and C), suggesting that the higher degree of CIN observed in SUM149PT cancer cells was functionally linked to the genesis of CD44 + /CD24 -/Low CSCs harboring amplified centrosomes.
Next we investigated the molecular mechanisms responsible for the development of centrosome abnormalities in CD44 + /CD24 -/Low CSCs. Because the oncoprotein cyclin E plays a key role in the development of centrosome abnormalities and is frequently overexpressed in TNBC tumors, we investigated the expression and activity of cyclin E in SUM149PT cells versus the CD44 + /CD24 -/Low subpopulation isolated by FACS sorting assay. Importantly, immunoblot analysis showed that cyclin E was overexpressed in the CD44 + /CD24 -/Low subpopulation compared to bulk SUM149PT cells (Fig. 2D). To investigate whether cyclin E overexpression was functionally linked to aberrant Cdk2 kinase activity, we analyzed the phosphorylation status of Rb tumor suppressor that is a downstream target of cyclin E/Cdk2 oncogenic signaling and its inactivation leads to centrosome aberrations (26). Higher phosphorylation and consequent inactivation of Rb was observed in the CD44 + /CD24 -/Low subpopulation compared to bulk SUM149PT cancer cells (Fig. 2D). Taken together these findings reveal that centrosome amplification was mechanistically linked to aberrant cyclin E/Cdk2 activity and consequent loss of Rb function in CD44 + /CD24 -/Low CSCs.
Because TNBC tumors display a poor prognosis that is associated to higher chemoresistance and stemness properties compared to luminal breast tumors, we investigated the extent to which inhibition of Cdk2 kinase activity selectively targeted the stem-like CD44 + /CD24 -/Low subpopulation and restored chemosensitivity of SUM149PT cancer cells. Bulk SUM149PT and CD44 + /CD24 -/Low cancer cells were treated with the anti-metabolite methotrexate (a conventional chemotherapeutic drug) or SU9516 (a small molecule inhibitor of Cdk2 activity) and cytotoxicity was quantified by analyzing the percentage of cells harboring cleaved PARP as a marker of apoptosis. Significantly, CD44 + /CD24 -/Low CSCs displayed a higher resistance to methotrexate while they where highly sensitive to the Cdk2 inhibitor SU9516 compared to bulk SUM149PT cancer cells (Fig. 3). Finally, we tested in vitro the translational therapeutic relevance of combining conventional anticancer agents with small molecule inhibitors of Cdk2 activity to restore chemosensitivity of TNBC tumors. SUM149PT cells were treated with paclitaxel, a taxane commonly employed in the treatment of TNBC tumors, alone or in combination with SU9516. Notably, our study revealed that combination of paclitaxel with SU9516 induced a stronger cytotoxic activity characterized by the majority of SUM149PT cells undergoing apoptosis compared to treatment with paclitaxel or SU9516 alone (Fig. 4).

Discussion
IBC tumors represent a rare and very aggressive subtype of breast carcinomas that display a TNBC phenotype defined as the absence of staining for ERα, PR and HER-2 receptors (1-3). TNBC tumors are insensitive to some of the most effective targeted therapies available for breast cancer treatment including endocrine therapies such as tamoxifen, aromatase inhibitors or fulvestrant and HER-2-directed therapy such as trastuzumab (27). To date, not a single targeted therapy has been approved for early-stage TNBC tumors and combination of conventional cytotoxic chemotherapeutic agents administered in a dose-dense or metronomic schedule remains the standard therapy (28). Significantly, a prospective analysis of 1,118 patients who received neoadjuvant chemotherapy at a single institution, of whom 255 (23%) had TNBC, found that patients with TNBC tumors had higher pathologic complete response (pCR) rates compared with non-TNBC patients (29). Because pCR is functionally linked to improved long-term outcomes, it is imperative to develop innovative targeted therapeutic strategies aimed to increase pCR rates with consequent benefits on the disease-free and overall survival of TNBC patients. Moreover, the use of anthracyclines (such as doxorubicin and epirubicin) and taxanes (such as paclitaxel and docetaxel) as chemotherapeutic agents for IBC tumors have been shown to improve outcomes (30). Although IBC tumors respond well initially to conventional chemotherapy, they often develop chemoresistance leading to tumor progression and poor outcome (31).
Development of EMT and consequent activation of stemness programming is responsible for invasion, tumor self-renewal and drug resistance leading to breast cancer progression, distant metastases and poor prognosis (32). For this reason the cancer stem cell-like phenotype commonly observed in IBC tumors may contribute to their aggressive nature but also may offer itself novel therapeutic strategies to the selective targeting of CD44 + /CD24 -CSCs from bulk tumors. Abrogation of BRCA1  and BRCA2 genes function can in part explain the high grade of CIN observed in inflammatory and non-inflammatory TNBC tumors resulting from centrosome amplification and impairment of the DNA repair machinery (10). Breast tumors carrying BRCA1 mutations are considered to be sensitive to inhibitors of poly (ADP-ribose) polymerases (PARPs) that are nuclear enzymes implicated in cellular responses to DNA injury provoked by genotoxic stress (33). PARP-1, the best characterized member of the PARP family, is essential to the repair of DNA single-strand breaks via the base excision repair pathway (34). Inhibitors of PARP-1 have been shown to enhance the cytotoxic effects of ionizing radiation and DNA-damaging chemotherapy agents, such as the methylating agents and topoisomerase II inhibitors (35). Moreover, recent studies suggests that PARP inhibitors could be used not only as chemo/radiotherapy sensitizers, but also as single agents to selectively kill cancers defective in DNA repair, specifically cancers with mutations in the breast cancer-associated genes BRCA1 and BRCA2 (36). Although clinical trials indicate that PARP-inhibitors have emerged as a promising new class of antineoplastic agents, significant numbers of TNBC tumors still recur (37). TNBC tumors recurrence following treatment with PARP-inhibitors can be explained by recent studies demonstrating that PARPinhibitors eliminate the bulk of tumor cells, but they have limited ability to eliminate CD44 + /CD24 -CSCs (38). To date no preclinical study has demonstrated the efficacy of PARPinhibitors in selective targeting of CD44 + /CD24 -CSCs in IBC tumors. For this reason the discovery of oncogenic signalings responsible for the maintenance and expansion of CD44 + /CD24 -CSCs is crucial for the development of innovative-targeted therapies aimed to drastically reduce the recurrence and poor outcome of IBC tumors.
The findings presented in this study demonstrate that IBC SUM149PT cancer cells exhibit higher CIN based on structural and numerical chromosome abnormalities than luminal ERα + MCF-7 cancer cells. Moreover, we established that only SUM149PT cancer cells contain a CD44 + /CD24 -/Low CSCs subpopulation displaying centrosome amplification that was functionally linked to cyclin E overexpression and Rb phosphorylation. Several studies have shown that elevated levels of cyclin E induce aberrant activation of Cdk2 kinase leading to Rb phosphorylation and inactivation with deleterious consequences on the control of centrosome duplication and CIN (14)(15)(16). Significantly, cyclin E is overexpressed in aggressive breast tumors and it has been associated with CIN, development of distant metastases and poor outcome (39). Concurring with our findings, it was previously demonstrated that SUM149PT cancer cells display very high levels of cyclin E expression for the duration of the cell cycle which is in contrast to cyclin E degradation observed in the mid to late S phase of normal cells. In addition, comparative genomic hybridization indicated that SUM149PT cells exhibit many chromosome copy number alterations, which may reflect prior or ongoing chromosome instability driven by cyclin E activity (40). Because we have demonstrated that cyclin E overexpression is restricted to the subpopulation of CD44 + /CD24 -CSCs isolated from SUM149PT cells, we treated bulk SUM149PT cells and CD44 + /CD24 -CSCs with a conventional anticancer drug (methotrexate) and a specific Cdk2 inhibitor (SU9516) to abrogate the cyclin E/Cdk2 oncogenic signaling. CSCs showed higher resistance to methotrexate than bulk SUM149PT cells, nonetheless CSCs were more sensitive to the cytotoxic effects of SU9516 indicating that molecular inhibition of Cdk2 kinase activity selectively targeted CSCs in IBC. Finally, we showed that combination of paclitaxel with SU9516 in vitro induces a stronger cytotoxic effect characterized by activation of apoptosis in SUM149PT cells. Importantly, the preclinical relevance of our findings is justified by recent studies demonstrating that administration of cyclin E siRNA in vivo inhibited breast tumor growth in nude mice. Moreover, the authors demonstrate that cyclin E siRNA synergistically enhanced the cell killing effects of chemotherapeutic agents in vitro and this combination greatly suppressed the tumor growth in nude mice. In conclusion our study demonstrate for the first time that aberrant activation of cyclin E/Cdk2 oncogenic signaling is restricted in CSCs derived from IBC cells and combination of conventional chemotherapy with small molecule inhibitors of Cdk2 kinase activity may represent a novel targeted therapeutic approach to treat aggressive IBC tumors with consequent benefits on the disease-free and overall survival of patients.