Na+/K+-ATPase α1 subunit,a novel therapeutic target for hepatocellular carcinoma

We aimed to identify the expression patterns of Na⁺/K⁺-ATPase (NKA) α subunits in human hepatocellular carcinoma (HCC) samples and evaluate these subunits as potential targets for HCC treatment. The mRNA expression profiles of NKA α subunits in human HCC samples were analyzed. We found that the mRNA expression for NKA α1 subunit (ATP1A1) was higher than that for other NKA α subunits. Also, ATP1A1 gene expression was markedly higher in HCC samples than in adjacent nontumor tissue samples. Western blotting data suggested that 6 of 14 (43%) HCC samples had elevated ATP1A1 protein expression. Furthermore, knockdown of ATP1A1 expression in human HCC HepG2 and MHCC97H cells markedly reduced their proliferation in vitro and suppressed the tumorigenicity of MHCC97H cells in vivo. Downregulation of ATP1A1 expression resulted in cell-cycle arrest at G2/M phase and apoptosis in HepG2 cells as well as decreased migration in Hep3B cells. We further validated that ATP1A1 downregulation caused intracellular accumulation of reactive oxygen species. Pretreatment with N-acetyl cysteine blocked cell-growth inhibition induced by ATP1A1 downregulation. Collectively, these data suggested that targeting ATP1A1 is a novel approach to the treatment of HCC.     

Sodium chloride (NaCl) potentiates digoxin-induced anti-tumor activity in small cell lung cancer

Small cell lung cancer (SCLC) is a malignant neuroendocrine tumor with very high mortality. Effective new therapy for advanced SCLC patients is urgently needed. By screening a FDA-approved drug library, we identified a cardiac glycoside (CG), namely digoxin (an inhibitor of cellular Na⁺/K⁺ ATPase pump), which was highly effective in inhibiting SCLC cell growth. Intriguing findings showed that NaCl supplement markedly enhanced the anti-tumor activities of digoxin in both in vitro and in vivo models of SCLC. Subsequent analysis revealed that this novel combination of digoxin/NaCl caused an up-regulation of intracellular Na⁺ and Ca²⁺ levels with an induction of higher resting membrane potential of SCLC cells. We also found that this combination lead to morphological shrinking of SCLC cells, together with high levels of cytochrome C release. Lastly, our data revealed that NaCl supplement was able to induce the expression of ATP1A1 (a Na⁺/K⁺ ATPase subunit), in which contributes directly to the increased sensitivity of SCLC cells to digoxin. Thus, this is the first demonstration that NaCl is a potent supplement necessitating superior anti-cancer effects of digoxin for SCLC. Further, our study suggests that digoxin treatment could need to be combined with NaCl supplement in future clinical trial of SCLC, particularly where low Na⁺ is often present in SCLC patients.     

FCY-302, a Novel Small Molecule,Induces Apoptosis in Leukemia and Myeloma Cells by Attenuating Key Antioxidant and Mitochondrial Enzymes

Arylidene analogs are well proven for biological activities. FCY-302, a novel small molecule belonging to this class, was screened for its biological efficacy in leukemia and myeloma cells. FCY-302 selectively inhibited proliferation of cancer cells with GI50 values of 395.2 nM, 514.6 Nm, and 642.4 nM in HL-60, Jurkat, and RPMI-8226 cells, respectively. The compound also increased sub-G0 peak in the cancer cell cycle and favored apoptosis determined by annexin V assay. The compound decreased the antiapoptotic Bcl-2 levels and increased proapoptotic Bax proteins in leukemia and myeloma cell lines. FCY-302 attenuated the mitochondrial membrane-bound Na⁺ /K⁺ ATPase, Ca²⁺ ATPase, and Mg²⁺ ATPase enzyme activities and significantly decreased activities of antioxidant enzymes like SOD, CAT, G_R, and GST in all the three cancer cells tested. Our findings suggest that FCY-302 inhibits the proliferation of leukemia and myeloma cancer cells by altering key mitochondrial and antioxidant enzymes, eventually driving them to apoptosis. These results drive focus on FCY-302 and its analogs to be developed as potential small molecules with bioactivities against cancer.     

IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA–NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ–STAT1 cascade augmented NK2R expression in CD8⁺ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic–polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8⁺ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8⁺ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8⁺ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8⁺ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8⁺ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.     

Sodium/potasium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides: a new paradigm for development of anti- breast cancer drugs?

Summary Prolonged exposure to 17β-estradiol (E₂) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERα and ERβ. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na⁺, K⁺-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na⁺, K⁺-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na⁺, K⁺-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na⁺, K⁺-ATPase inhibitors and ER antagonists.     

Role of Na+, K+-ATPase α1 subunit in the intracellular accumulation of cisplatin

The present study was undertaken to identify what regulates intracellular cisplatin (CDDP) accumulation and what changes in membrane fraction of CDDDP-resistant cell line. The CDDP-resistant rat hepatoma cell line, H4-II-E/CDDP, shows a significant decrease in intracellular platinum accumulation compared with parental H4-II-E cells, although there was no difference in the efflux of CDDP between these two cell lines. In this study, we examined the contribution of functional change in active transport to the CDDP resistance of H4-II-E/CDDP cells. Compared with the resistant cells, platinum accumulation in the parental cells was clearly decreased by low temperature or ATP depletion. In addition, the Na⁺, K⁺-ATPase inhibitor ouabain and the K⁺ channel inhibitor tetraethylammonium decreased platinum accumulation in parental cells but did not change the accumulation in resistant cells. Amphotericin B, an antifungal agent, increased the intracellular platinum accumulation in resistant cells to the same level as in parent cells. Western blot analysis demonstrated that the Na⁺, K⁺-ATPase α₁ subunit was reduced in resistant cells compared with the parental cells, although there was no difference in the expression of the β1 subunit between the two cell lines. Furthermore, the Na⁺, K⁺-ATPase α1 subunit of H4-II-E was decreased following a 24-h exposure to CDDP. These results suggest that Na⁺, K⁺-ATPase-dependent active transport of CDDP does not occur in resistant cells, and, furthermore, our findings provide the first evidence that the Na⁺, K⁺-ATPase α1 subunit plays an important role in the transport of CDDP.     

Extracellular galectin-3 programs multidrug resistance through Na+/K+-ATPase and P-glycoprotein signaling

Galectin-3 (Gal-3, LGALS3) is a pleotropic versatile, 29–35 kDa chimeric gene product, and involved in diverse physiological and pathological processes, including cell growth, homeostasis, apoptosis, pre-mRNA splicing, cell-cell and cell-matrix adhesion, cellular polarity, motility, adhesion, activation, differentiation, transformation, signaling, regulation of innate/adaptive immunity, and angiogenesis. In multiple diseases, it was found that the level of circulating Gal-3 is markedly elevated, suggesting that Gal-3-dependent function is mediated by specific interaction with yet an unknown ubiquitous cell-surface protein. Recently, we showed that Gal-3 attenuated drug-induced apoptosis, which is one of the mechanisms underlying multidrug resistance (MDR). Here, we document that MDR could be mediated by Gal-3 interaction with the house-keeping gene product e.g., Na⁺/K⁺-ATPase, and P-glycoprotein (P-gp). Gal-3 interacts with Na⁺/K⁺-ATPase and induces the phosphorylation of P-gp. We also find that Gal-3 binds P-gp and enhances its ATPase activity. Furthermore Gal-3 antagonist suppresses this interaction and results in a decrease of the phosphorylation and the ATPase activity of P-gp, leading to an increased sensitivity to doxorubicin-mediated cell death. Taken together, these findings may explain the reported roles of Gal-3 in diverse diseases and suggest that a combined therapy of inhibitors of Na⁺/K⁺-ATPase and Gal-3, and a disease specific drug(s) might be superior to a single therapeutic modality.     

Substance P activates responses correlated with tumour growth in human glioma cell lines bearing tachykinin NK1 receptors

The neuropeptide substance P (SP), by stimulating tachykinin NK₁receptors (NK₁R), triggers a number of biological responses in human glioma cells which are potentially relevant for tumour growth. First, radioligand binding studies demonstrated the presence of tachykinin NK₁R on SNB-19, DBTRG-05 MG and U373 MG, but not on U138 MG and MOG-G-GCM human glioma cell lines. Second, application of SP or neurokinin A (NKA) to NK₁R⁺glioma cell lines increased the secretion of interleukin 6 (IL-6) and potentiated IL-6 secretion induced by IL-1β. SP also up-regulated the release of transforming growth factor β1 (TGF-β1) by the U373 MG glioma cell line. Third, SP induced new DNA synthesis and enhanced the proliferation rate of NK₁R⁺, but not of NK₁R⁻glioma cell lines. Also, NKA stimulated the proliferation and cytokine secretion in NK₁R⁺glioma cell lines. All the stimulant effects of SP/NKA on NK₁R⁺glioma cell lines were completely blocked by a specific tachykinin NK₁R antagonist, MEN 11467. These data support the potential use of tachykinin NK₁R antagonist for controlling the proliferative rate of human gliomas. © 1999 Cancer Research Campaign     

Inhibition of platelet-derived growth factor receptor synergistically increases the pharmacological effect of tamoxifen in estrogen receptor α positive breast cancer

The platelet-derived growth factor (PDGF) family, a complex and imperative group of proangiogenic factors, acts as strong cell growth chemokines and is essential for the progression of malignancy in humans. In the present study, it was observed that aberrant PDGFB expression is associated with survival rates in patients with estrogen receptor-positive (ER⁺) breast cancer unlike other subtypes, including PDGFA, PDGFC and PDGFD. Accordingly, the effect of specific PDGF receptor (PDGFR) inhibitors on ER-α⁺ breast cancer cells was investigated. To block the PDGF-BB signaling pathway, PDGFR inhibitors (sunitinib or ponatinib) were employed. Sunitinib and ponatinib were found to arrest the cell cycle at the G0-G1 phase. In addition, the two PDGFR inhibitors were revealed to significantly inhibit cell growth and decrease the expression of matrix metalloproteinase-1, which is one of the metastasis-related genes. Finally, the combined effects of the two PDGFR inhibitors with tamoxifen were investigated. The results demonstrated that the combination of two PDGFR inhibitors with tamoxifen inhibited the growth of cells more consistently, compared with the effect mediated by tamoxifen alone. Therefore, it is proposed that PDGFR inhibitors, including sunitinib and ponatinib, should be applied effectively to treat ER-α⁺ breast cancer.     

Activation of ASC induces apoptosis or necrosis,depending on the cell type,and causes tumor eradication

The adaptor protein ASC (also called TMS1) links certain NLR proteins (e.g., NLRC4, NLRP3) and caspases. It is involved in the chemosensitivity of tumor cells and inflammation. Here, we found that ASC activation using NLRC4 mimicry or an autoinflammatory disease‐associated NLRP3 mutant induced necrosis in COLO205 colon adenocarcinoma cells, but induced caspase‐8‐dependent apoptosis in NUGC‐4 stomach cancer cells. As the Fas ligand induced caspase‐8‐dependent apoptosis in COLO205 cells, caspase‐8 was intact in this cell line. ASC‐mediated necrosis was preceded by lysosomal leakage, and diminished by inhibitors for vacuolar H⁺‐ATPase, cathepsins, and calpains but not by inhibitors for caspase‐8, or aspartic proteases, suggesting that lysosomes and certain proteases were involved in this process. Finally, growing tumors of transplanted human cancer cells in nude mice were eradicated by the activation of endogenous ASC in the tumor cells, irrespective of the form of cell death. Thus, ASC mediates distinct forms of cell death in different cell types, and is a promising target for cancer therapy. (Cancer Sci 2010)     

The Sodium Pump α1 Subunit as a Potential Target to Combat Apoptosis-Resistant Glioblastomas

Purpose

To review the involvement of the ion transporter Na⁺/K⁺-ATPase (NaK) in the migration and proliferation of glioma cells. Preliminary studies indicate that NaK α₁ subunits seem to be upregulated in a proportion of glioblastomas but not in normal brain tissues.

Design

The present review focuses on (1) the natural resistance of migrating malignant glioma cells to apoptosis, (2) autophagic cell death as an alternative to combat malignant gliomas, (3) the fact that reducing the levels of malignant glioma cell motility can restore proapoptotic drug sensitivity, and (4) on the observation that inhibiting the NaK activity reduces both glioma cell proliferation and migration.

Results

The natural ligands of the NaK are the cardiotonic steroids. A hemisynthetic derivative of 2″-oxovoruscharin (UNBS1450), a novel cardenolide, displays unique structural features, making its binding affinity to NaK α subunits (including α₁) 10 to 100 times higher than that of other cardenolides. UNBS1450 markedly decreases intracellular ATP concentration in glioma cells, disorganizes the actin cytoskeleton, and leads to autophagic cell death in NaK α₁ over-expressing glioma cells.

Conclusions

Glioblastoma patients who do not respond to chemotherapy and whose tumors over-express NaK α₁ subunits could benefit from a treatment using ligands with marked binding affinity for the NaK α₁ subunit.     

Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24⁺CD44⁺ and ALDH⁺ tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for further investigation of this combination in PDA.     

Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy

The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H⁺ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H⁺ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.     

Epi-reevesioside F inhibits Na+/K+-ATPase,causing cytosolic acidification,Bak activation and apoptosis in glioblastoma

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC₅₀ values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na⁺/K⁺-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na⁺ but not Ca²⁺, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na⁺/K⁺-ATPase, leading to overload of intracellular Na⁺ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.     

Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation

Background:

Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored.

Methods:

Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit.

Results:

We show that despite similar reovirus replication in p53^+/+ and p53^−/− cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53^−/− or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors.

Conclusion:

Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.     

Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy

Recombinant Newcastle disease virus (rNDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. In this study, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of inserting both interleukin-2 (IL-2) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) delivered by rNDV. We demonstrated that rNDV expressing TRAIL (rNDV-TRAIL) or both human IL-2 and TRAIL (rNDV-IL-2-TRAIL) significantly enhanced inherent anti-neoplastic of rNDV by inducing apoptosis. And we showed that apoptosis-related genes mRNA expression was increased after treated with rNDV-TRAIL or rNDV-IL-2-TRAIL compared with rNDV and rNDV-IL-2. We also demonstrated that both rNDV-IL-2 and rNDV-IL-2-TRAIL induced proliferation of the CD4⁺ and CD8⁺ in treated mice and elicited expression of TNF-α and IFN-γ antitumor cytokines. These mice treated with oncolytic agents exhibited significant reduction in tumor development compared with mice treated with the parental virus. In addition, experiments in both hepatocellular carcinoma and melanoma-bearing mice demonstrated that the genetically engineered rNDV-IL-2-TRAIL exhibited prolonged animals’ survival compared with rNDV, rNDV-IL-2, and rNDV-TRAIL. In conclusion, the immunotherapy and oncolytic virotherapy properties of NDV can be enhanced by the introduction of IL-2 and TRAIL genes, whose products initiated a broad cascade of immunological affects and induced tumor cells apoptosis in the microenvironment of the immune system.     

CD19+CD24hiCD38hiBregs involved in downregulate helper T cells and upregulate regulatory T cells in gastric cancer

Regulatory B cells (Bregs) play a critical role in inflammation and autoimmune disease. We characterized the role of Bregs in the progression of gastric cancer. We detected an increase in Bregs producing IL-10 both in peripheral blood mononuclear cells (PBMCs) and in gastric tumors. Multicolor flow cytometry analysis revealed that a subset of CD19⁺CD24^hiCD38^hi B cells produces IL-10. Functional studies indicated that increased Bregs do not inhibit the proliferation of CD3⁺T cells or CD4⁺ helper T cells (Th cells). However, Bregs do suppress the secretion of IFN-γ and TNF-α by CD4⁺Th cells. CD19⁺CD24^hiCD38^hiBregs were also found to correlate positively with CD4⁺FoxP3⁺ regulatory T cells (Tregs). Neutralization experiments showed that Bregs convert CD4⁺CD25⁻ effector T cells to CD4⁺FoxP3⁺Tregs via TGF-β1. Collectively, these findings demonstrate that increased Bregs play a immunosuppressive role in gastric cancer by inhibiting T cells cytokines as well as conversion to Tregs. These results may provide new clues about the underlying mechanisms of immune escape in gastric cancer.     

Distinct breast cancer stem/progenitor cell populations require either HIF1α or loss of PHD3 to expand under hypoxic conditions

The heterogeneous nature of breast cancer is a result of intrinsic tumor complexity and also of the tumor microenvironment, which is known to be hypoxic. We found that hypoxia expands different breast stem/progenitor cell populations (cells with increased aldehyde dehydrogenase activity (Aldefluor⁺), high mammosphere formation capacity and CD44⁺CD24^−/low cells) both in primary normal epithelial and tumor cells. The presence of the estrogen receptor (ER) limits hypoxia-dependent CD44⁺CD24^−/low cell expansion. We further show that the hypoxia-driven cancer stem-like cell enrichment results from a dedifferentiation process. The enhanced mammosphere formation and Aldefluor⁺ cell content observed in breast cancer cells relies on hypoxia-inducible factor 1α (HIF1α). In contrast, the CD44⁺CD24^−/low population expansion is HIF1α independent and requires prolyl hydroxylase 3 (PHD3) downregulation, which mimics hypoxic conditions, leading to reduced CD24 expression through activation of NFkB signaling. These studies show that hypoxic conditions expand CSC populations through distinct molecular mechanisms. Thus, potential therapies that combine current treatments for breast cancer with drugs that target CSC should take into account the heterogeneity of the CSC subpopulations.