Salinomycin Promotes Anoikis and Decreases the CD44+/CD24- Stem-Like Population via Inhibition of STAT3 Activation in MDA-MB-231 Cells

Triple-negative breast cancer (TNBC) is an aggressive tumor subtype with an enriched CD44⁺/CD24^- stem-like population. Salinomycin is an antibiotic that has been shown to target cancer stem cells (CSC); however, the mechanisms of action involved have not been well characterized. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in MDA-MB-231 breast cancer cells. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44⁺/CD24^- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, interleukin-6 (IL-6)-induced STAT3 activation was strongly suppressed by salinomycin challenge. These findings support the notion that salinomycin may be potentially efficacious for targeting breast cancer stem-like cells through the inhibition of STAT3 activation.     

Anti‐EGFR antibody‐drug conjugate for triple‐negative breast cancer therapy

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR⁺ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.     

Spectrum of EGFR Gene Copy Number Changes and KRAS Gene Mutation Status in Korean Triple Negative Breast Cancer Patients

Anti-epidermal growth factor receptor (EGFR) therapy has been tried in triple negative breast cancer (TNBC) patients without evaluation of molecular and clinical predictors in several randomized clinical studies. Only fewer than 20% of metastatic TNBCs showed response to anti-EGFR therapy. In order to increase the overall response rate, first step would be to classify TNBC into good or poor responders according to oncogenic mutation profiles. This study provides the molecular characteristics of TNBCs including EGFR gene copy number changes and mutation status of EGFR and KRAS gene in Korean TNBC patients. Mutation analysis for EGFR, KRAS, BRAF and TP53 from a total of 105 TNBC tissue samples was performed by direct sequencing, peptide nucleic acid-mediated PCR clamping method and real-time PCR. Copy number changes of EGFR gene were evaluated using multiplex ligation-dependent probe amplification. Out of all 105 TNBCs, 15.2% (16/105) showed EGFR copy number changes. Among them, increased or decreased EGFR copy number was detected in 13 (5 single copy gain, 2 amplification and 4 high-copy number amplification) and 3 cases (3 hemizygous deletion), respectively. The mutation frequencies of KRAS, EGFR and TP53 gene were 1.9% (G12V and G12D), 1.0% (exon 19 del) and 31.4%, respectively. There was no BRAF V600E mutation found. Future studies are needed to evaluate the clinical outcomes of TNBC patients who undergo anti-EGFR therapy according to the genetic status of EGFR.     

Targeting the EGFR/PCNA Signaling Suppresses Tumor Growth of Triple-Negative Breast Cancer Cells with Cell-Penetrating PCNA Peptides

Tyrosine 211 (Y211) phosphorylation of proliferation cell nuclear antigen (PCNA) coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant cells, both nuclear EGFR (nEGFR) expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC). Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP) inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP), which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC.     

MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

Introduction

Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method

In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results

MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions

MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.     

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.     

Discovery and SAR studies of novel 2-anilinopyrimidine-based selective inhibitors against triple-negative breast cancer cell line MDA-MB-468

Triple-negative breast cancers (TNBCs) are characterized as an invasive and intractable subtype of breast cancers. Overexpression of epidermal growth factor receptor (EGFR) has been considered to be an important target for TNBC therapy, but efficacies of EGFR inhibitors in clinical trials are elusive. In this study, novel series of 2-anilinopyrimidines were synthesized in an effort to identify selective inhibitors against an EGFR-overexpressing TNBC cell line. Biological evaluation demonstrated that compounds 21 and 38, with a 4-methylpiperidine group and a high ClogP value, exhibited good potency and selectivity for the TNBC cell line. This study has provided evidence to support further development of 2-anilinopyrimidine-based TNBC selective inhibitors and investigation of the targets of compounds 21 and 38.     

RAD6B is a major mediator of triple negative breast cancer cisplatin resistance: Regulation of translesion synthesis/Fanconi anemia crosstalk and BRCA1 independence

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with few therapy options besides chemotherapy. Although platinum-based drugs have shown initial activity in BRCA1-mutated TNBCs, chemoresistance remains a challenge. Here we show that RAD6B (UBE2B), a principal mediator of translesion synthesis (TLS), is overexpressed in BRCA1 wild-type and mutant TNBCs, and RAD6B overexpression correlates with poor survival. Pretreatment with a RAD6-selective inhibitor, SMI#9, enhanced cisplatin chemosensitivity of BRCA1 wild-type and mutant TNBCs. SMI#9 attenuated cisplatin-induced PCNA monoubiquitination (TLS marker), FANCD2 (Fanconi anemia (FA) activation marker), and TLS polymerase POL η. SMI#9-induced decreases in γH2AX levels were associated with concomitant inhibition of H2AX monoubiquitination, suggesting a key role for RAD6 in modulating cisplatin-induced γH2AX via H2AX monoubiquitination. Concordantly, SMI#9 inhibited γH2AX, POL η and FANCD2 foci formation. RAD51 foci formation was unaffected by SMI#9, however, its recruitment to double-strand breaks was inhibited. Using the DR-GFP-based assay, we showed that RAD6B silencing or SMI#9 treatment impairs homologous recombination (HR) in HR-proficient cells. DNA fiber assays confirmed that restart of cisplatin-stalled replicating forks is inhibited by SMI#9 in both BRCA1 wild-type and mutant TNBC cells. Consistent with the in vitro data, SMI#9 and cisplatin combination treatment inhibited BRCA1 wild-type and mutant TNBC growth as compared to controls. These RAD6B activities are unaffected by BRCA1 status of TNBCs suggesting that the RAD6B function in TLS/FA crosstalk could occur in HR-dependent and independent modes. Collectively, these data implicate RAD6 as an important therapeutic target for TNBCs irrespective of their BRCA1 status.     

Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.     

Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple‐negative breast cancer cells

Triple‐negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti‐proliferative effect in cell lines of the basal‐like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI‐103 combination significantly reduced the level of both phospho‐AKT and phospho‐ERK in two susceptible BL subtype cell lines, SUM149PT and MDA‐MB‐468, whereas it had little or no effect on the level of phospho‐ERK in two non‐susceptible cell lines (HS578T and MDA‐MB‐231) of mesenchymal stem‐like (MSL) TNBC subtype. The gefitinib/PI‐103 combination also significantly induced caspase‐3/7‐mediated PARP cleavage and reduced two anti‐apoptotic proteins, XIAP and Bcl‐2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl‐1) protein was markedly decreased by gefitinib/PI‐103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.     

Hypoxia-induced tRNA-derived fragments,novel regulatory factor for doxorubicin resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of epithelial breast malignancy, and chemoresistance is the major obstacle for cancer therapy. TNBC is associated with a hypoxic phenotype, and hypoxia contributes to the chemoresistance in breast cancer. Transfer RNA-derived fragments (tDRs) represent a new class of small noncoding RNAs that can be induced specifically by hypoxia. Here, we conducted a comparative analysis of the aberrant expression of tDRs in hypoxia-treated TNBC cell lines through the use of high-throughput sequencing technique. Quantitative real-time polymerase chain reaction was used to validate the differently expressed tDRs between two samples. The results showed that tDR-0009 [derived from transfer RNA (tRNA)^Gly-GCC-1-1] and tDR-7336 (derived from tRNA ^{Gly-GCC-1–2}) were significantly upregulated when the SUM-1315 cell lines were stimulated by hypoxia. Gene ontology (GO) and pathway analysis indicated that these two upregulated tDRs were mainly involved in maintenance of stem cell population and cellular response to interleukin (IL)-6, which may be the underlying mechanism of hypoxia-induced tDRs that facilitate the doxorubicin resistance in TNBC. The protein–protein interaction network for predicted target genes established by the STRING database manifested that tDR-0009 (tDR-7336) might be involved in the chemoresistance of TNBC via regulation of the activation of phosphorylation of STAT3. In summary, our study provided a comprehensive analysis of the deviant expression profiling of tDRs in hypoxia-treated TNBC cell lines. Specific tDRs may be a new class of regulatory factors involved in the hypoxia-induced chemoresistance in TNBC, and they could serve as potential biomarkers and intervention targets.