Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1-/- mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK's effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment

HOXC10 expression supports the development of chemotherapy resistance by fine tuning DNA repair in breast cancer cells

Development of drug resistance is a major factor limiting the continued success of cancer chemotherapy. To overcome drug resistance, understanding the underlying mechanism(s) is essential. We found that HOXC10 is overexpressed in primary carcinomas of the breast, and even more significantly in distant metastasis arising after failed chemotherapy. High HOXC10 expression correlates with shorter recurrence-free and overall survival in patients with estrogen receptor-negative breast cancer undergoing chemotherapy. We found that HOXC10 promotes survival in cells treated with doxorubicin, paclitaxel, or carboplatin by suppressing apoptosis and upregulating NF-κB. Overexpressed HOXC10 increases S-phase-specific DNA damage repair by homologous recombination (HR) and checkpoint recovery in cells at three important phases. For double-strand break repair, HOXC10 recruits HR proteins at sites of DNA damage. It enhances resection and lastly, it resolves stalled replication forks, leading to initiation of DNA replication following DNA damage. We show that HOXC10 facilitates, but is not directly involved in DNA damage repair mediated by HR. HOXC10 achieves integration of these functions by binding to, and activating cyclin-dependent kinase, CDK7, which regulates transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II. Consistent with these findings, inhibitors of CDK7 reverse HOXC10-mediated drug resistance in cultured cells. Blocking HOXC10 function, therefore, presents a promising new strategy to overcome chemotherapy resistance in breast cancer. ©2016 AACR

HOXA5 determines cell fate transition and impedes tumor initiation and progression in breast cancer through regulation of E-cadherin and CD24

10.1038/onc.2016.95ONCOGENE35425539-555

Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3.

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1-/- mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNKs effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment

Enhancing the abscopal effect of radiation and immune checkpoint inhibitor therapies with magnetic nanoparticle hyperthermia in a model of metastatic breast cancer

Purpose: Enhancing immune responses in triple negative breast cancers (TNBCs) remains a challenge. Our study aimed to determine whether magnetic iron oxide nanoparticle (MION) hyperthermia (HT) can enhance abscopal effects with radiotherapy (RT) and immune checkpoint inhibitors (IT) in a metastatic TNBC model.Methods: One week after implanting 4T1-luc cells into the mammary glands of BALB/c mice, tumors were treated with RT (3 × 8 Gy)±local HT, mild (HTM, 43 °C/20 min) or partially ablative (HTAbl, 45 °C/5 min plus 43 °C/15 min),±IT with anti-PD-1 and anti-CTLA-4 antibodies (both 4 × 10 mg/kg, i.p.). Tumor growth was measured daily. Two weeks after treatment, lungs and livers were harvested for histopathology evaluation of metastases.Results: Compared to untreated controls, all treatment groups demonstrated a decreased tumor volume; however, when compared against surgical resection, only RT + HTM+IT, RT + HTAbl+IT and RT + HTAbl had similar or smaller tumors. These cohorts showed more infiltration of CD3+ T-lymphocytes into the primary tumor. Tumor growth effects were partially reversed with T-cell depletion. Combinations that proved most effective for primary tumors generated modest reductions in numbers of lung metastases. Conversely, numbers of lung metastases showed potential to increase following HT + IT treatment, particularly when compared to RT. Compared to untreated controls, there was no improvement in survival with any treatment.Conclusions: Single-fraction MION HT added to RT + IT improved local tumor control and recruitment of CD3+ T-lymphocytes, with only a modest effect to reduce lung metastases and no improvement in overall survival. HT + IT showed potential to increase metastatic dissemination to lungs

Increased uptake of doxorubicin by cells undergoing heat stress does not explain its synergistic cytotoxicity with hyperthermia

Purpose: A proposed mechanism for the enhanced effectiveness of hyperthermia and doxorubicin (Dox) combinations is increased intracellular Dox concentrations resulting from heat-induced cell stress. The purpose of this study was to determine whether specific varied Dox and heat combinations produce measurable effects greater than the additive combination, and whether these effects can be attributed to heat-induced increases in intracellular Dox concentrations. Methods: HCT116, HT29 and CT26 cells were exposed to Dox and water bath heating independently. A clonogenic survival assay was used to determine cell killing and intracellular Dox concentrations were measured in HCT116 cells with mass spectrometry. Cells were exposed to heating at 42 °C (60 min) and 0.5 µg/ml of Dox at varying intervals. Synergy was determined by curve-fitting and isobologram analysis. Results: All cell lines displayed synergistic effects of combined heating and Dox. A maximum synergistic effect was achieved with simultaneous cell exposure to Dox and heat. For exposures at 42 °C, the synergistic effect was most pronounced at Dox concentrations <0.5 µg/ml. Increased intracellular concentrations of Dox in HCT116 cells caused by heat-stress did not generate a concomitant thermal enhancement. Conclusions: Simultaneous exposure of HCT116 cells to heating and Dox is more effective than sequential exposure. Heat-induced cell responses are accompanied by increased intracellular Dox concentrations; however, clonogenic survival data do not support this as the cause for synergistic cytotoxicity