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.
The production, secretion, and localization of surface proteins of hepatitis B virus (HBV) and the ratio of large to small surface protein S was studied in HepG2 cells transfected with the wild-type and mutant pre-S1 and pre-S2/S promoters of HBV molecular clones 313.1 (GenBank accession no. AY161147) and 761.1 (GenBank accession no. AY161159) from two patients with occult HBV infection. Fusion constructs were made by in frame fusion of the wild-type surface gene to the mutant pre-S1 and pre-S2/S promoters and wild-type promoter so that the structural part of the small surface protein remains identical. HepG2 cells transfected transiently were used for analysis. HBV surface proteins production and secretion was determined by enzyme linked immuno assay (ELISA) and localization by immunofluorescence. Immunoprecipitation of the large, middle, and small surface protein was carried out in transient transfected and metabolically labeled cells to determine the ratio of the large to small surface protein. The results indicate that HepG2 cells transfected with mutant HBV promoters had reduced HBV surface proteins secretion compared to wild-type HBV. HepG2 cells transfected with mutant HBV pre-S1 and pre-S2/S promoters showed cytoplasmic aggregation of HBV surface proteins compared to wild-type HBV promoters, which showed diffuse cytoplasmic localization. In all cases, the HBV surface proteins localized to the endoplasmic reticulum. The ratio between the large and small surface protein was 1.89 and 0.56 with mutant HBV 313.1 and 761.1 pre-S1 and pre-S2/S promoters, respectively, compared to 0.17 in wild-type. Thus, the aggregation of surface proteins, altered ratio and secretion of surface proteins were possibly the causes of occult hepatitis B infection.
The C-terminus of the HIV-1 reverse transcriptase heterodimer was reconstructed into a single polypeptide. The construct encodes the p51 thumb (T) and connection (C) subdomains joined through a linker region to the p66 connection (C) and RNase H (R) domain. The TCCR protein was purified from insoluble fractions of Escherichia coli lysates. The TCCR construct maintains Mn(2+)-dependent RNase H activity and specifically cleaves the substrate mimicking the tRNA removal required for second-strand transfer reactions.
Introduction: Dedifferentiation involves terminally differentiated cells reverting back to less differentiated cells such as precursor cells or stem cells within their own lineage allowing the cells to proliferate again before re-differentiation. Tumorigenesis involves uncontrolled cell proliferation and many host-related factors aid in dedifferentiation of tumor cells into stem cell like phenotypes hence providing a mechanism leading to tumor heterogeneity and aggressiveness. Pluripotency factors Oct4, Nanog and Sox2 have been implicated in dedifferentiation and are highly expressed in tumors. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our laboratory demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit pluripotency related transcription factors and reverse the process of dedifferentiation associated with uncontrolled cell proliferation in tumorigenesis. Results: Here, we provide evidence that HNK inhibits the ability of breast cancer cells to form mammospheres. HNK treatment inhibits dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 in MCF-7, MDA-MB-231, T47D, MDA-MB-468 breast cancer cells. An analysis of underlying signaling mechanism reveal that HNK inhibits phosphorylation of Stat3 and the downregulation of pluripotency factors is mediated via inhibition of Stat3. Stat3 inhibitor, Stattic potentiates the effect of HNK while overexpression of constitutively active Stat3 interferes with HNK-mediated inhibition of Oct4, Nanog and Sox2. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we show that HNK-mediated inhibition of Oct4, Nanog and Sox2 is regulated by tumor suppressor LKB1. The ability of HNK to inhibit mammosphere formation is abrograted in breast cancer cells stably-silenced for LKB1. In vitro and in vivo analyses show novel functional interactions between HNK, Stat3, LKB1 and dedifferentiation/pluripotency markers. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 via tumor suppressor LKB1-Stat3 axis in inhibiting “dedifferentiation” in breast cancer. Citation Format: Sonali Sengupta, Michael Y. Bonner, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Inhibiting “Dedifferentiation” in breast cancer cells using Honokiol - a plant derived polyphenol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-61. doi:10.1158/1538-7445.AM2014-LB-61
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