Inhibition of IGF1R signaling abrogates resistance to afatinib (BIBW2992) in EGFR T790M mutant lung cancer cells.

Non-small cell lung cancer (NSCLC) patients with an epidermal growth factor receptor (EGFR) mutation have benefited from treatment of reversible EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Acquisition of a secondary mutation in EGFR T790M is the most common mechanism of resistance to first generation EGFR TKIs, resulting in therapeutic failure. Afatinib is a second generation of EGFR TKI that showed great efficacy against tumors bearing the EGFR T790M mutation, but it failed to show the improvement on overall survival of lung cancer patients with EGFR mutations possibly because of novel acquired resistance mechanisms. Currently, there are no therapeutic options available for lung cancer patients who develop acquired resistance to afatinib. To identify novel resistance mechanism(s) to afatinib, we developed afatinib resistant cell lines from a parental human-derived NSCLC cell line, H1975, harboring both EGFR L858R and T790M mutations. We found that activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway contributes to afatinib resistance in NSCLC cells harboring the T790M mutation. IGF1R knockdown not only significantly sensitizes resistant cells to afatinib, but also induces apoptosis in afatinib resistance cells. In addition, combination treatment with afatinib and linsitinib shows more than additive effects on tumor growth in in vivo H1975 xenograft. Therefore, these finding suggest that IGF1R inhibition or combination of EGFR-IGF1R inhibition strategies would be potential ways to prevent or potentiate the effects of current therapeutic options to lung cancer patients demonstrating resistance to either first or second generation EGFR TKIs.

Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer.

Forkhead box protein A1 (FOXA1) is essential for androgen-dependent prostate cancer (PCa) growth. However, how FOXA1 levels are regulated remains elusive and its therapeutic targeting proven challenging. Here, we report FOXA1 as a nonhistone substrate of enhancer of zeste homolog 2 (EZH2), which methylates FOXA1 at lysine-295. This methylation is recognized by WD40 repeat protein BUB3, which subsequently recruits ubiquitin-specific protease 7 (USP7) to remove ubiquitination and enhance FOXA1 protein stability. They functionally converge in regulating cell cycle genes and promoting PCa growth. FOXA1 is a major therapeutic target of the inhibitors of EZH2 methyltransferase activities in PCa. FOXA1-driven PCa growth can be effectively mitigated by EZH2 enzymatic inhibitors, either alone or in combination with USP7 inhibitors. Together, our study reports EZH2-catalyzed methylation as a key mechanism to FOXA1 protein stability, which may be leveraged to enhance therapeutic targeting of PCa using enzymatic EZH2 inhibitors.

Targeting FOXA1-mediated repression of TGF-ß signaling suppresses castration-resistant prostate cancer progression.

Prostate cancer (PC) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation antiandrogen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and a basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable upregulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of the TGF-ß pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of the TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 downregulation induces TGF-ß signaling, EMT, and cell motility, which is effectively blocked by the TGF-ß receptor I inhibitor galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-ß signaling, indicated by SMAD2 phosphorylation, in CRPC as compared with primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PC cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-ß signaling, and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.

Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis.

Lung cancer often has a poor prognosis, with brain metastases a major reason for mortality. We modified lonidamine (LND), an antiglycolytic drug with limited efficacy, to mitochondria-targeted mito-lonidamine (Mito-LND) which is 100-fold more potent. Mito-LND, a tumor-selective inhibitor of oxidative phosphorylation, inhibits mitochondrial bioenergetics in lung cancer cells and mitigates lung cancer cell viability, growth, progression, and metastasis of lung cancer xenografts in mice. Mito-LND blocks lung tumor development and brain metastasis by inhibiting mitochondrial bioenergetics, stimulating the formation of reactive oxygen species, oxidizing mitochondrial peroxiredoxin, inactivating AKT/mTOR/p70S6K signaling, and inducing autophagic cell death in lung cancer cells. Mito-LND causes no toxicity in mice even when administered for eight weeks at 50 times the effective cancer inhibitory dose. Collectively, these findings show that mitochondrial targeting of LND is a promising therapeutic approach for investigating the role of autophagy in mitigating lung cancer development and brain metastasis.

Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator.

Enhancer of Zeste 2 (EZH2) is the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which catalyzes histone H3 lysine 27 trimethylation (H3K27me3) at target promoters for gene silencing. Here, we report that EZH2 activates androgen receptor (AR) gene transcription through direct occupancy at its promoter. Importantly, this activating role of EZH2 is independent of PRC2 and its methyltransferase activities. Genome-wide assays revealed extensive EZH2 occupancy at promoters marked by either H3K27ac or H3K27me3, leading to gene activation or repression, respectively. Last, we demonstrate enhanced efficacy of enzymatic EZH2 inhibitors when used in combination with AR antagonists in blocking the dual roles of EZH2 and suppressing prostate cancer progression in vitro and in vivo. Taken together, our study reports EZH2 as a transcriptional activator, a key target of which is AR, and suggests a drug-combinatory approach to treat advanced prostate cancer.

Mitochondria-Targeted Honokiol Confers a Striking Inhibitory Effect on Lung Cancer via Inhibiting Complex I Activity.

We synthesized a mitochondria-targeted honokiol (Mito-HNK) that facilitates its mitochondrial accumulation; this dramatically increases its potency and efficacy against highly metastatic lung cancer lines in vitro, and in orthotopic lung tumor xenografts and brain metastases in vivo. Mito-HNK is >100-fold more potent than HNK in inhibiting cell proliferation, inhibiting mitochondrial complex ?, stimulating reactive oxygen species generation, oxidizing mitochondrial peroxiredoxin-3, and suppressing the phosphorylation of mitoSTAT3. Within lung cancer brain metastases in mice, Mito-HNK induced the mediators of cell death and decreased the pathways that support invasion and proliferation. In contrast, in the non-malignant stroma, Mito-HNK suppressed pathways that support metastatic lesions, including those involved in inflammation and angiogenesis. Mito-HNK showed no toxicity and targets the metabolic vulnerabilities of primary and metastatic lung cancers. Its pronounced anti-invasive and anti-metastatic effects in the brain are particularly intriguing given the paucity of treatment options for such patients either alone or in combination with standard chemotherapeutics.

Honokiol Decreases Lung Cancer Metastasis through Inhibition of the STAT3 Signaling Pathway.

Lung cancer is the leading cause of cancer death in the United States. Metastasis to lymph nodes and distal organs, especially brain, leads to severe complications and death. Preventing lung cancer development and metastases is an important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in the extracts of magnolia bark, has a favorable bioavailability profile and recently has been shown to readily cross the blood-brain barrier. In the current study, we evaluated the antimetastatic effects of HNK in both the lymph node and brain mouse models of lung tumor metastasis. We tested the efficacy of HNK in preventing 18 H2030-BrM3 cell (brain-seeking human lung tumor cells) migration to lymph node or brain. In an orthotopic mouse model, HNK significantly decreased lung tumor growth compared with the vehicle control group. HNK also significantly reduced the incidence of lymph node metastasis and the weight of mediastinal lymph nodes. In a brain metastasis model, HNK inhibits metastasis of lung cancer cells to the brain to approximately one third of that observed in control mice. We analyzed HNK's mechanism of action, which indicated that its effect is mediated primarily by inhibiting the STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation irrespective of the mutation status of EGFR, and knockdown of STAT3 abrogated both the antiproliferative and the antimetastatic effects of HNK. These observations suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer metastasis. Cancer Prev Res; 10(2); 133-41. ©2016 AACR.

Honokiol targets mitochondria to halt cancer progression and metastasis.

Cancer continues to be the leading cause of death worldwide. Plants have a long history of use in the treatment of cancer. Honokiol (HNK) is an important bioactive compound found in the bark of Magnolia tree, and has been shown to inhibit cancer growth and metastasis in many cell types in vitro and in animal models. Resistance to chemotherapy and radiotherapy is the major obstacle for cure of cancer. Combination of HNK with many traditional chemotherapeutic drugs as well as radiation sensitizes cancer cells to apoptotic death, suggesting that HNK not only directly inhibits primary cancers and metastasis, but also has potential to overcome drug resistance. Ultimately, this may mean that HNK could be combined with traditional chemotherapies administered at lower doses to significantly reduce toxicity, meanwhile enhance efficacy. As a natural compound, HNK is composed of polyphenols and has been described in many studies targeting multiple key cell signaling molecules. Mitochondria are the main hub for cellular energy production and play an important role in cell survival, and are the key target identified for HNK to mediate cancer cell death, survival, and metastasis. In this review, we have summarized different aspects of HNK's anti-cancer effects from recent accumulated literature, as well as the underlying molecular mechanisms. This review is primarily focused on the effects of HNK on epidermal growth factor receptor (EGFR) and signal transduction and activator of transcription 3 (STAT3) signaling, as well as the broader regulation of mitochondrial function and cancer cell metabolism.