Yunfang Li

Lats2 , a putative tumor suppressor, inhibits G1/S transition

Lats2 is a new member of the Lats tumor suppressor family. The human LATS2 gene is located at chromosome 13q11–12, which has been shown to be a hot spot (67%) for LOH in nonsmall cell lung cancer. In order to understand the function of LATS2 in the control of tumor development, we ectopically expressed mouse Lats2 via retroviral infection in NIH3T3/v-ras cells to examine whether Lats2 plays a role in suppressing tumor development and regulating cell proliferation. We have found that ectopic expression of Lats2 in NIH3T3/v-ras cells suppresses development of tumors in athymic nude mice and inhibits proliferation of NIH3T3/v-ras cells in an in vitro assay. Cell cycle profile analysis demonstrated that ectopic expression of Lats2 inhibited the G1/S transition. Further mechanistic studies revealed that cyclin E/CDK2 kinase activity was downregulated in Lats2-transduced NIH3T3/v-ras cells, while other cell cycle regulators controlling the G1/S transition were not affected. We have also shown that LATS2 kinase activity and two LATS conserved domains (LCDs) are required for Lats2 to suppress tumorigenicity and to inhibit cell growth. In addition, the LATS2 protein is cytoplasmic during interphase in NIH3T3 cells, while it becomes localized to the mitotic apparatus during mitosis. Finally, we propose a model in which a combination of mammalian Lats2 and Lats1 control cell proliferation by negatively regulating different cell cycle check points.

COX-2 inhibitor celecoxib prevents chronic morphine-induced promotion of angiogenesis, tumour growth, metastasis and mortality, without compromising analgesia.

Morphine and its congener opioids are the main therapy for severe pain in cancer. However, chronic morphine treatment stimulates angiogenesis and tumour growth in mice. We examined if celecoxib (a cyclooxygenase-2 (COX-2) inhibitor) prevents morphine-induced tumour growth without compromising analgesia. The effect of chronic treatment with celecoxib (by gavage) and/or morphine (subcutaneously), or PBS on tumour prostaglandin E2 (PGE2), COX-2, angiogenesis, tumour growth, metastasis, pain behaviour and survival was determined in a highly invasive SCK breast cancer model in A/J mice. Two weeks of chronic morphine treatment at clinically relevant doses stimulates COX-2 and PGE2 (4.5-fold compared to vehicle alone) and angiogenesis in breast tumours in mice. This is accompanied by increased tumour weight (∼35%) and increased metastasis and reduced survival. Co-administration of celecoxib prevents these morphine-induced effects. In addition, morphine and celecoxib together provided better analgesia than either agent alone. Celecoxib prevents morphine-induced stimulation of COX-2, PGE2, angiogenesis, tumour growth, metastasis and mortality without compromising analgesia in a murine breast cancer model. In fact, the combination provided significantly better analgesia than with morphine or celecoxib alone. Clinical trials of this combination for analgesia in chronic and severe pain in cancer are warranted.

LATS1 tumor suppressor regulates G2/M transition and apoptosis

The LATS1 gene is a mammalian member of the novel lats tumor suppressor family. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumors. Our previous studies have shown that inactivation of Drosophila lats leads to up-regulation of cyclin A in the fly, and the human LATS1 protein associates with CDC2 in early mitosis in HeLa cells, suggesting that the lats gene family may negatively regulate cell proliferation by modulating CDC2/Cyclin A activity. We demonstrate here that transduction of the human breast cancer cell MCF-7 with recombinant LATS1 adenovirus (Ad-LATS1), but not with EGFP adenovirus (Ad-EGFP), inhibits in vitro cell proliferation. Ectopic expression of LATS1 in MCF-7 cells specifically down-regulates Cyclin A and Cyclin B protein levels and dramatically reduces CDC2 kinase activity, leading to a G2/M blockade. Furthermore, Ad-LATS1 suppresses anchorage-independent growth of MCF-7 cells in soft agar and tumor formation in athymic nude mice. We also demonstrate that ectopic expression of LATS1 in MCF-7 cells and human lung cancer cell H460 up-regulates the level of BAX proteins and induces apoptosis. Finally, we show that LATS1 kinase activity is required for its ability to inhibit cell growth and induce apoptosis. The results indicate that the LATS1 tumor suppressor may play an important role in the control of human tumor development and that LATS1 suppresses tumorigenesis by negatively regulating cell proliferation and modulating cell survival.

Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids

Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of mu opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.

Morphine-Induced Epidermal Growth Factor Pathway Activation in Non-Small Cell Lung Cancer

BACKGROUND: Epidermal growth factor receptor (EGFR) is coactivated by the &mgr;-opioid receptor (MOR), expressed on non–small cell lung cancer (NSCLC) cells and human lung cancer. We hypothesized that clinically used opioid analgesics that are MOR agonists coactivate EGFR, resulting in growth- and survival-promoting signaling. METHODS: We used H2009, a human adenocarcinoma NSCLC cell line, with constitutive EGFR phosphorylation, which showed increased expression of MOR and the &dgr;-opioid receptor by reverse transcriptase polymerase chain reaction. We used Western immunoblotting, magnetic bead–based Bio-Plex cytokine assay, immunofluorescent staining, BrdU incorporation enzyme-linked immunosorbent assay, and BioCoat™ Matrigel™ invasion assay to examine cell signaling, cytokine expression, colocalization of MOR and EGFR in human lung cancer, and cell proliferation and invasion, respectively. RESULTS: Similar to epidermal growth factor (EGF), morphine stimulated phosphorylation of EGFR, Akt/protein kinase B (Akt), and mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) signaling in H2009 cells. Opioid receptor (OR) antagonist, naloxone, EGFR tyrosine kinase inhibitor, erlotinib, and silencing of MOR and &dgr;-opioid receptor abrogated morphine- and EGF-induced phosphorylation of signaling, suggestive of OR-mediated coactivation of EGFR. H2009 cells secreted significantly higher levels of cytokines compared with control Beas2B epithelial cells. H2009-conditioned medium stimulated MOR expression in Beas2B cells, suggesting that cytokines secreted by H2009 may be associated with increased OR expression in H2009. We observed colocalization of EGFR and MOR, in human NSCLC tissue. Functionally, morphine- and EGF-induced proliferation and invasion of H2009 cells was ameliorated by naloxone as well as erlotinib. CONCLUSION: Morphine-induced phosphorylation of EGFR occurs via ORs, leading to downstream MAPK/ERK, Akt phosphorylation, cell proliferation, and increased invasion. Notably, ORs are also associated with EGF-induced phosphorylation of EGFR. Increased coexpression of MOR and EGFR in human lung cancer suggests that morphine may have a growth-promoting effect in lung cancer.

Morphine stimulates cancer progression and mast cell activation and impairs survival in transgenic mice with breast cancer

BACKGROUND Morphine stimulates angiogenesis and cancer progression in mice. We investigated whether morphine influences tumour onset, development, and animal model survival, and whether µ-opioid receptor (MOR), lymphangiogenesis, mast cell activation, and substance P (SP) are associated with the tumour-promoting effects of morphine. METHODS Transgenic mice with a rat C3(1) simian virus 40 large tumour antigen fusion gene which demonstrate the developmental spectrum of human infiltrating ductal breast carcinoma were used. Mice were treated at different ages with clinically relevant doses of morphine or phosphate-buffered saline to determine the effect on tumour development and progression, and on mouse survival. Tumours were analysed for MOR, angiogenesis, lymphangiogenesis, SP, and mast cell activation by immunofluorescent- or laser scanning confocal-microscopy. Cytokine and SP levels were determined by enzyme-linked immunosorbent assay. RESULTS Morphine did not influence tumour development when given before the onset of tumour appearance, but significantly promoted progression of established tumours, and reduced survival. MOR-immunoreactivity (ir) was observed in larger but not in smaller tumours. Morphine treatment resulted in increased tumour angiogenesis, peri-tumoural lymphangiogenesis, mast cell activation, and higher levels of cytokines and SP in tumours. SP-ir co-localized with mast cells and elsewhere in the tumours. CONCLUSIONS Morphine does not affect the onset of tumour development, but it promotes growth of existing tumours, and reduces overall survival in mice. MOR may be associated with morphine-induced cancer progression, resulting in shorter survival. Mast cell activation by morphine may contribute to increased cytokine and SP levels, leading to cancer progression and refractory pain.

NADPH oxidase contributes to coronary endothelial dysfunction in the failing heart.

Increased reactive oxygen species (ROS) produced by the failing heart can react with nitric oxide (NO), thereby decreasing NO bioavailability. This study tested the hypothesis that increased ROS generation contributes to coronary endothelial dysfunction in the failing heart. Congestive heart failure (CHF) was produced in six dogs by ventricular pacing at 240 beats/min for 4 wk. Studies were performed at rest and during treadmill exercise under control conditions and after treatment with the NADPH oxidase inhibitor and antioxidant apocynin (4 mg/kg iv). Apocynin caused no significant changes in heart rate, aortic pressure, left ventricular (LV) systolic pressure, LV end-diastolic pressure, or maximum rate of LV pressure increase at rest or during exercise in normal or CHF dogs. Apocynin caused no change in coronary blood flow (CBF) in normal dogs but increased CBF at rest and during exercise in animals with CHF (P < 0.05). Intracoronary ACh caused dose-dependent increases of CBF that were blunted in CHF. Apocynin had no effect on the response to ACh in normal dogs but augmented the response to ACh in CHF dogs (P < 0.05). The oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal were significantly greater in failing than in normal myocardium. Furthermore, coelenterazine chemiluminescence for O(2)(-) was more than twice normal in failing myocardium, and this difference was abolished by apocynin. Western blot analysis of myocardial lysates demonstrated that the p47(phox) and p22(phox) subunits of NADPH were significantly increased in the failing hearts, while real-time PCR demonstrated that Nox2 mRNA was significantly increased. The data indicate that increased ROS generation in the failing heart is associated with coronary endothelial dysfunction and suggest that NADPH oxidase may contribute to this abnormality.

Ecto-5′-Nucleotidase Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction

This study examined whether endogenous extracellular adenosine acts to facilitate the adaptive response of the heart to chronic systolic overload. To examine whether endogenous extracellular adenosine can protect the heart against pressure-overload–induced heart failure, transverse aortic constriction was performed on mice deficient in extracellular adenosine production as the result of genetic deletion of CD73. Although there was no difference in left ventricular size or function between CD73-deficient mice (knockout [KO] mice) and wild-type mice under unstressed conditions, aortic constriction for 2 or 4 weeks induced significantly more myocardial hypertrophy, left ventricular dilation, and left ventricular dysfunction in KO mice compared with wild-type mice. Thus, after 2 weeks of transverse aortic constriction, left ventricular fractional shortening decreased to 27.4±2.5% and 21.9±1.7% in wild-type and KO mice, respectively (P<0.05). Consistent with a role of adenosine in reducing tissue remodeling, KO mice displayed increased myocardial fibrosis and myocyte hypertrophy compared with wild-type mice. Furthermore, adenosine treatment reduced phenylephrine-induced cardiac myocyte hypertrophy and collagen production in cultured neonatal rat cardiac myocytes and cardiac fibroblasts, respectively. Consistent with a role for adenosine in modulating cardiomyocyte hypertrophy, KO mice demonstrated increased activation of mammalian target of rapamycin signaling, accompanied by higher expression of the hypertrophy marker atrial natriuretic peptide. Conversely, the adenosine analogue 2-chloro-adenosine significantly reduced cell size, mammalian target of rapamycin/p70 ribosomal S6 kinase activation, and atrial natriuretic peptide expression in cultured neonatal cardiomyocytes. These data demonstrate that CD73 helps to preserve cardiac function during chronic systolic overload by preventing maladaptive tissue remodeling.

Overexpression of Mcl-1 Confers Multidrug Resistance, Whereas Topoisomerase IIβ Downregulation Introduces Mitoxantrone-Specific Drug Resistance in Acute Myeloid Leukemia

Drug resistance is a serious challenge in cancer treatment and can be acquired through multiple mechanisms. These molecular changes may introduce varied extents of resistance to different therapies and need to be characterized for optimal therapy choice. A recently discovered small molecule, ethyl-2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate) (CXL017), reveals selective cytotoxicity toward drug-resistant leukemia. A drug-resistant acute myeloid leukemia cell line, HL60/MX2, also failed to acquire resistance to CXL017 upon chronic exposure and regained sensitivity toward standard therapies. In this study, we investigated the mechanisms responsible for HL60/MX2 cells’ drug resistance and the molecular basis for its resensitization. Results show that the HL60/MX2 cell line has an elevated level of Mcl-1 protein relative to the parental cell line, HL60, and its resensitized cell line, HL60/MX2/CXL017, whereas it has a reduced level of topoisomerase IIβ. Mcl-1 overexpression in HL60/MX2 cells is mainly regulated through phospho-extracellular signal-regulated protein kinases 1 and 2–mediated Mcl-1 stabilization, whereas the reduction of topoisomerase IIβ in HL60/MX2 cells is controlled through genetic downregulation. Upregulating Mcl-1 introduces multidrug resistance to standard therapies, whereas its downregulation results in significant cell death. Downregulating topoisomerase IIβ confers resistance specifically to mitoxantrone, not to other topoisomerase II inhibitors. Overall, these data suggest that Mcl-1 overexpression is a critical determinant for cross-resistance to standard therapies, whereas topoisomerase IIβ downregulation is specific to mitoxantrone resistance.

Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017): a novel scaffold that resensitizes multidrug resistant leukemia cells to chemotherapy.

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer, and there is a pressing need for new therapies. We have recently developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017), derived from a dual inhibitor of Bcl-2 and SERCA proteins, sHA 14-1, with selective cytotoxicity toward MDR cancer cell lines in vitro. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights toward its preferential targeting of drug-resistant cancer. CXL017 selectively suppressed the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, even after chronic exposure to CXL017, HL60/MX2 failed to develop stable resistance to CXL017, whereas it acquired >2000-fold resistance to cytarabine (Ara-C), the major first-line chemotherapy for the treatment of acute myeloid leukemia (AML). Remarkably, instead of acquiring further cross-resistance, HL60/MX2 cells exposed to CXL017 were resensitized to standard therapies (10- to 100-fold). Western blotting analyses revealed that CXL017 exposure significantly down-regulated Mcl-1 and Bax and up-regulated Noxa, Bim, Bcl-X(L), SERCA2, and SERCA3 proteins, along with a reduction in endoplasmic reticulum (ER) calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the down-regulation of Mcl-1 and the up-regulation of Noxa and Bim along with the decrease in ER calcium content are likely responsible for CXL017-induced resensitization of MDR cancer cells. These data also demonstrate the unique potential of CXL017 to overcome MDR in cancer treatment.