Nitric Oxide Induces Cell Death by Regulating Anti-Apoptotic BCL-2 Family Members

Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim^−/−/Puma^−/− MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death.     

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax^−/−Bak^−/− cells and better than either Bid^−/− or dominant-negative caspase-9-expressing cells. Only Bim^−/− and Bax^−/−Bak^−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid^−/− cells, it readily did so in Bim^−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1^−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X_L with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.     

Poldip2 Knockout Results in Perinatal Lethality,Reduced Cellular Growth and Increased Autophagy of Mouse Embryonic Fibroblasts

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2−/− embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2−/− mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2−/− MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2−/− cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2−/− MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2−/− cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21^CIP1 is increased in passage 4/5 Poldip2−/− MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.     

Breast cancer dependence on MCL-1 is due to its canonical anti-apoptotic function

High levels of the anti-apoptotic BCL-2 family member MCL-1 are frequently found in breast cancer and, appropriately, BH3-mimetic drugs that specifically target MCL-1’s function in apoptosis are in development as anti-cancer therapy. MCL-1 also has reported non-canonical roles that may be relevant in its tumour-promoting effect. Here we investigate the role of MCL-1 in clinically relevant breast cancer models and address whether the canonical role of MCL-1 in apoptosis, which can be targeted using BH3-mimetic drugs, is the major function for MCL-1 in breast cancer. We show that MCL-1 is essential in established tumours with genetic deletion inducing tumour regression and inhibition with the MCL-1-specific BH3-mimetic drug {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 significantly impeding tumour growth. Importantly, we found that the anti-tumour functions achieved by MCL-1 deletion or inhibition were completely dependent on pro-apoptotic BAX/BAK. Interestingly, we find that MCL-1 is also critical for stem cell activity in human breast cancer cells and high MCL1 expression correlates with stemness markers in tumours. This strongly supports the idea that the key function of MCL-1 in breast cancer is through its anti-apoptotic function. This has important implications for the future use of MCL-1-specific BH3-mimetic drugs in breast cancer treatment.Subject terms: Cancer models, Cell biology, Genetics     

Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

The Cip/Kip family, namely, p21^Cip1, p27^Kip1, and p57^Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21^Cip1 and p27^Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2^−/− p21^−/− p27^−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57^Kip2 downregulation in the absence of p21^Cip1 and p27^Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21^−/− p27^−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2^−/− p21^−/− p27^−/− survivors displayed all phenotypes described for p27^−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.     

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.     

Consequences of the combined loss of BOK and BAK or BOK and BAX

The multi-BCL-2 homology domain pro-apoptotic BCL-2 family members BAK and BAX have critical roles in apoptosis. They are essential for mitochondrial outer-membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome-c, which promote activation of the caspase cascade and cellular demolition. The BOK protein has extensive amino-acid sequence similarity to BAK and BAX and is expressed in diverse cell types, particularly those of the female reproductive tissues. The BOK-deficient mice have no readily discernible abnormalities, and its function therefore remains unresolved. We hypothesized that BOK may exert functions that overlap with those of BAK and/or BAX and examined this by generating Bok^−/−Bak^−/− and Bok^−/−Bax^−/− mice. Combined loss of BOK and BAK did not elicit any noticeable defects, although it remains possible that BOK and BAK have critical roles in developmental cell death that overlap with those of BAX. In most tissues examined, loss of BOK did not exacerbate the abnormalities caused by loss of BAX, such as defects in spermatogenesis or the increase in neuronal populations in the brain and retina. Notably, however, old Bok^−/−Bax^−/− females had abnormally increased numbers of oocytes from different stages of development, indicating that BOK may have a pro-apoptotic function overlapping with that of BAX in age-related follicular atresia.     

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.     

Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells

CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage⁻Sca-1⁺KIT⁺ (LSK) cells decreased in the fetal liver of CALM ^−/− mice. Also, colony forming activity was impaired in CALM^−/− LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM^−/− LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM ^−/− murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM ^−/− MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM ^−/− MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM ^−/− MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM ^−/− MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM ^−/− KIT⁺ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.     

Anti-Proliferative Effects of Evodiamine on Human Breast Cancer Cells

Endocrine sensitivity, assessed by the expression of estrogen receptor (ER), has long been the predict factor to guide therapeutic decisions. Tamoxifen has been the most successful hormonal treatment in endocrine-sensitive breast cancer. However, in estrogen-insensitive cancer tamoxifen showed less effectiveness than in estrogen-sensitive cancer. It is interesting to develop new drugs against both hormone-sensitive and insensitive tumor. In this present study we examined anticancer effects of evodiamine extracted from the Chinese herb, Evodiae fructus, in estrogen-dependent and –independent human breast cancer cells, MCF-7 and MDA-MB-231 cells, respectively. Evodiamine inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner with concentration of 1×10⁻⁶ and 1×10⁻⁵ M. Evodiamine also induced apoptosis via up-regulation of caspase 7 activation, PARP cleavage (Bik and Bax expression). The expression of ER α and β in protein and mRNA levels was down-regulated by evodiamine according to data from immunoblotting and RT-PCR analysis. Overall, our results indicate that evodiamine mediates degradation of ER and induces caspase-dependent pathway leading to inhibit proliferation of breast cancer cell lines. It suggests that evodiamine may in part mediate through ER-inhibitory pathway to inhibit breast cancer cell proliferation.     

Nucleostemin delays cellular senescence and negatively regulates TRF1 protein stability

Nucleostemin (NS) encodes a nucleolar GTP-binding protein highly enriched in the stem cells and cancer cells. To determine its biological activity in vivo, we generated NS loss- and gain-of-function mouse models. The embryogenesis of homozygous NS-null (NS^−/−) mice was aborted before the blastula stage. Although the growth and fertility of heterozygous NS-null (NS^+/−) mice appeared normal, NS^+/− mouse embryonic fibroblasts (MEFs) had fewer NS proteins, a lower population growth rate, and higher percentages of senescent cells from passage 5 (P5) to P7 than their wild-type littermates. Conversely, transgenic overexpression of NS could rescue the NS^−/− embryo in a dose-dependent manner, increase the population growth rate, and reduce the senescent percentage of MEFs. Cell cycle analyses revealed increased pre-G₁ percentages in the late-passage NS^+/− MEF cultures compared to the wild-type cultures. We demonstrated that NS could interact with telomeric repeat-binding factor 1 (TRF1) and enhance the degradation but not the ubiquitination of the TRF1 protein, which negatively regulates telomere length and is essential for early embryogenesis. This work demonstrates the roles of NS in establishing early embryogenesis and delaying cellular senescence of MEFs and reveals a mechanism of a NS-regulated degradation of TRF1.     

Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 is generated in endosomes by an endogenously or ascorbate-induced S-nitrosothiol-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid β (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We showed by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as ³⁵S labeling experiments that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (WT MEFs), Tg2576 MEFs, and N2a neuroblastoma cells but not in APP^−/− and APLP2^−/− MEFs. Transfection of APP^−/− cells with a vector encoding APP restored nuclear import of anMan-containing HS. In WT MEFs and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEFs, the β-inhibitor blocked transport, but the γ-inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of WT MEFs. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine, indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.     

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg^−/− mouse ES cells and human cancer cell lines. Parg^−/− mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg^−/− mouse ES cells. Parg^−/− ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.     

Involvement of p53 and p21 in Cellular Defects and Tumorigenesis in Atm−/− Mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm^−/− p53^−/− mice develop lymphomas earlier than Atm^−/− or p53^−/− mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G₁/S checkpoint is abolished in Atm^−/− p53^−/− mouse embryonic fibroblasts (MEFs) following γ-irradiation, suggesting that the partial G₁ cell cycle arrest in Atm^−/− cells following γ-irradiation is due to the residual p53 response in these cells. In addition, the Atm^−/− p21^−/− MEFs are more severely defective in their cell cycle G₁ arrest following γ-irradiation than Atm^−/− and p21^−/− MEFs. The Atm^−/− MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm^−/− p21^−/− MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm^−/− p53^−/− MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm^−/− cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm^−/− MEFs is lower than that in Atm^+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm^−/− MEFs is likely due to increased stability of the p21 protein.     

Promyelocytic Leukemia (PML) Protein Plays Important Roles in Regulating Cell Adhesion,Morphology, Proliferation and Migration

PML protein plays important roles in regulating cellular homeostasis. It forms PML nuclear bodies (PML-NBs) that act like nuclear relay stations and participate in many cellular functions. In this study, we have examined the proteome of mouse embryonic fibroblasts (MEFs) derived from normal (PML^+/+) and PML knockout (PML^−/−) mice. The aim was to identify proteins that were differentially expressed when MEFs were incapable of producing PML. Using comparative proteomics, total protein were extracted from PML^−/− and PML^+/+ MEFs, resolved by two dimensional electrophoresis (2-DE) gels and the differentially expressed proteins identified by LC-ESI-MS/MS. Nine proteins (PML, NDRG1, CACYBP, CFL1, RSU1, TRIO, CTRO, ANXA4 and UBE2M) were determined to be down-regulated in PML^−/− MEFs. In contrast, ten proteins (CIAPIN1, FAM50A, SUMO2 HSPB1 NSFL1C, PCBP2, YWHAG, STMN1, TPD52L2 and PDAP1) were found up-regulated. Many of these differentially expressed proteins play crucial roles in cell adhesion, migration, morphology and cytokinesis. The protein profiles explain why PML^−/− and PML^+/+ MEFs were morphologically different. In addition, we demonstrated PML^−/− MEFs were less adhesive, proliferated more extensively and migrated significantly slower than PML^+/+ MEFs. NDRG1, a protein that was down-regulated in PML^−/− MEFs, was selected for further investigation. We determined that silencing NDRG1expression in PML^+/+ MEFs increased cell proliferation and inhibited PML expression. Since NDRG expression was suppressed in PML^−/− MEFs, this may explain why these cells proliferate more extensively than PML^+/+ MEFs. Furthermore, silencing NDRG1expression also impaired TGF-β1 signaling by inhibiting SMAD3 phosphorylation.     

Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.