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Metastasis and drug resistance are the major causes of mortality in patients with non–small cell lung cancer (NSCLC). Several receptor tyrosine kinases (RTKs), including AXL, are involved in the progression of NSCLC. The AXL/MER/SKY subfamily is involved in cell adhesion, motility, angiogenesis, and signal transduction and may play a significant role in the invasiveness of cancer cells. Notably, no specific inhibitors of AXL have been described. A series of CL1 sublines with progressive invasiveness established from a patient with NSCLC has been identified that positively correlates with AXL expression and resistance to chemotherapeutic drugs. The ectopic overexpression of AXL results in elevated cell invasiveness and drug resistance. Nuclear factor-κB (NF-κB) signaling activity is associated with AXL expression and may play an important role in the enhancement of invasiveness and doxorubicin resistance, as shown by using the NF-κB inhibitor, sulfasalazine, and IκB dominant-negative transfectants. In the current study, sulfasalazine exerted a synergistic anticancer effect with doxorubicin and suppressed cancer cell invasiveness in parallel in CL1 sublines and various AXL-expressing cancer cell lines. Phosphorylation of AXL and other RTKs (ErbB2 and epidermal growth factor receptor) was abolished by sulfasalazine within 15 min, suggesting that the inhibition of NF-κB and the kinase activity of RTKs are involved in the pharmacologic effects of sulfasalazine. Our study suggests that AXL is involved in NSCLC metastasis and drug resistance and may therefore provide a molecular basis for RTK-targeted therapy using sulfasalazine to enhance the efficacy of chemotherapy in NSCLC. [Cancer Res 2007;67(8):3878–87]

Supplementary Figures 1-6 from Sulfasalazine Suppresses Drug Resistance and Invasiveness of Lung Adenocarcinoma Cells Expressing AXL

Background: Pancreatic adenocarcinoma (PDAC) is known for its deregulated TGFβ signal pathway. Therapeutic strategy targeting TGFβ is controversial due to the dual role of TGFβ with anti-proliferation in early phase and pro-metastatic in late phase of cancer progression. KLF10, an early response gene of TGFβ, positively feedback the antiproliferative effect in cancer. Recent studies revealed KLF10 expression were suppressed epigenetically in various cancers including PDAC. In contrary to TGFβ, KLF10 transcriptionally suppress Slug and Sirtuin 6 to prevent metastasis in advanced PDAC. The role of KLF10 in regulating tumorigenesis and stem cell phenotype is still unknown. Materials and Methods: From 105 patients of resectable PDAC, KLF10 expression level was evaluated to be reduced in 62% of tumor specimens. Low KLF10 expression correlated with larger tumor size and rapid loco-regional recurrence. In murine model of Kras mutation (KC mice), additional depletion of KLF10 induced 57% of PDAC compared to 0% at 18 to 24 wk of age. PDAC cells were genetically manipulated with KLF10mRNA silencing (PDACshKLF10) or inducible overexpression (PDACoeKLF10). Using limiting dilution assay of tumor growth, and orthotopic tumor implantation, we found higher tumorigenic ability of PDACshKlf10 compared to that of wild type. The stem cell phenotypes of PDACshKLF10 and PDACoeKLF10 were confirmed by sphere formation assay and FACS analysis of surface markers including CD24/CD44, ESA/c-Met, CD47 and CD133. Results: Notch signal pathway was found to be significantly enhanced from microarray analysis of wild type versus PDACshKLF10 cells. The increased Notch signal molecules were confirmed in RNA and protein level in genetically manipulated PDAC cells as well as clinical tissue specimens. Using Chip-PCR and luciferase promoter assay, KLF10 was demonstrated to bind to the promoter of Notch receptor 1, 3, and 4. DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a Notch inhibitor, suppressed in vitro spheroid formation, cell surface expression of stem cell markers, and tumor growth of PDACshKLF10 in orthotopic murine model. We conclude that KLF10 modulates stem cell phenotype of PDAC by transcriptionally suppressing Notch signal pathway. Loss of KLF10 in PDAC patients leads to Notch signal activation, development of stem cell phenotype and tumor progression. Notch inhibition may reverse malignant growth of PDAC with reduced KLF10 expression. Conflict of interest statement: nothing to declare Citation Format: Yi-Chih Tsai, Su-Liang Chen, Shu-Ling Peng, Kuang-Hung Cheng, Shih-Sheng Jiang, Shuang-En Chuang, Ch'ang Hui-Ju. Krupple like factor 10 modulates stem cell phenotype of pancreatic adenocarcinoma via transcriptional regulation of Notch signal pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2001.

MicroRNAs (miRNAs) play important roles in various biologic processes, and aberrant miRNA expression is one of the mechanisms of tumorigenesis. Previous studies indicate that oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a and contributes to the development of cervical cancer. In addition, the traditional Chinese medicine Gynostemma pentaphyllum (Thunb.) Makino may exert its pharmacologic effects through modifying the gut microbiota by regulating hepatic miR-34a expression. MiR-449 miRs are members of the tumor suppressor miR-34 family and are evolutionarily conserved. In this study, we demonstrated that, in lung cancer cells, ectopic expression of miR-449 family, including miR-449a, miR-449b, and miR-449c, decreased the levels of several G1 phase proteins including cyclin D1, cyclin D3, CDK4, and CDK6 via directly targeting their mRNA’s 3’-UTR. We also observed a differential induction of apoptosis vs. senescence by the miR-449 cluster. While miR-449a and miR-449c induced senescence, miR-449b induced apoptosis and sensitized cancer cells to apoptotic stimuli, such as serum deprivation. Interestingly, among this cluster miRNAs, only miR-449a could induce significant senescence-associated secretory phenotype (SASP), e.g., expression of IL-8, IL-6, IL-1b, and TNF-alpha. Moreover, we also found that miR-449a increased NF-kB phosphorylation at S276, but not phosphorylation site of S536, and promoted the expression of IL-8 mRNA. In this regard, the gut microbiota has been indicated to induce proinflammatory cytokines such as IL-8 to influence the outcomes of cancer patients. In addition to MRX34 (a miR-34a mimic) used in clinical trials, miR-449 family may also potentially become a new therapeutic candidate for NSCLC and other solid tumors’ treatment. Citation Format: Chun-Yu Cho, Shih-Ying Chung, Shuang-En Chuang. Differential regulation of apoptosis and senescence by the miR-449 family microRNAs and its implications in tumor microenvironments of NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A29.

The anticancer effects of honokiol, an active component isolated from Chinese traditional herb Magnolia officinalis, had been noticed in recent decade. It had been reported to induce apoptosis, anti-agiogenesis and chemo/radio-sensitization on cancer cells. However, its effects on autophagy induction and cancer stem cell elimination have not yet been investigated. To more evaluate the potential of honokiol in cancer therapy, we explored its effects on these two critical events in glioblastoma multiforme (GBM) DBTRG-05MG cells. The honokiol-induced autophagy was evidenced by the time and dose-dependent increase of LC3-II in treated cells. During this autophagy induction, the phosphorylated AMP-dependent kinase alpha was dose-dependently increased in accompany with the decrease of phosphorylated mTOR protein level. In addition, the phosphorylated p38 was also markedly increased parallel with the elevation of LC3-II. Further investigation on the role of this MAPK p38 pathway in honokiol-induced autophagy is ongoing. In accordance with the growth inhibition and massive cell death induced by high dose of honokiol (50 μM), the phsphorylated Akt and Rb were dramatically suppressed in treated cells. As it is suggested that autophagy is a protective response of cancer cell to environmental stress, an autophagy inhibitor hydroxychloroquine was therefore co-administrated to enhance the effects of honokiol against GBM cells. Significantly, hydroxychloroquine augmented the honokiol-induced cell death, indicating the crucial role of autophagy in honokiol-induced anticancer effects. Moreover, honokiol appeared to have effects on the elimination of cancer stem-like cells. By UV laser-equipped flow cytometer, a small percentage of cancer stem-like side population (SP) cells was detected and sorted from another GBM cell line (GBM 8401). The expressions of stemness genes such as CD133, nestin, Oct4, NOTCH3, IHH and SMO in SP cells were much higher than those in non-SP cells. Treatment with honokiol (5 μM) for 48h decreased the percentage of GBM 8401 SP cells from 1.5% down to 0.2%. These results suggested the potential role of honokiol in GBM treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3778. doi:10.1158/1538-7445.AM2011-3778

Leukemia is a serious disease that affects both children and adults. Although all-trans retinoic acid (ATRA) and targeted drug such as imatinib could effectively treat acute promyelocytic (APL) and chronic myelogenous leukemia (CML), respectively, rare therapeutics is available when drug resistance or recurrence occurred. Searching for alternative agent for refractory patient is urgently needed. Our previous studies had found that a human urine extract CDA-2 could induce differentiation of HL60 APL cells as well as potentiate the effects of ATRA and imatinib on HL60 and K562 CML cells, respectively. Purification of active compound possessing these anti-leukemic effects from CDA-2 was thus performed. Recently, an active compound named as P18.9 was isolated. It induced differentiation of K562 CML cells as evidenced by the nitroblue-tetrazolium (NBT) reduction test. At higher dose, flow cytometry analysis showed that P18.9 induced sub-G1 apoptotic fraction of K562 cells and enhanced the sub-G1 fraction induced by imatinib at a scale of about two folds. Although P18.9 did not induce differentiation of HL60 APL cells, it substantially enhanced the ATRA-induced differentiation. This is the first study to isolate an active compound from human urine, which exerts anti-leukemic effects alone or cooperatively with clinical used drugs. The chemical structure of P18.9 had been identified and further subsequent structure modification is ongoing. Continuing the effort to develop P18.9 and its derivatives as novel therapeutics for refractory APL or CML patients is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C189.