LKB1 represses focal adhesion kinase (FAK) signaling via a FAK-LKB1 complex to regulate FAK site maturation and directional persistence.

Liver kinase ß1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr(397)/Tyr(861) and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr(397)-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.

STE20-related kinase adaptor protein α (STRADα) regulates cell polarity and invasion through PAK1 signaling in LKB1-null cells.

LKB1 is a Ser/Thr kinase, and its activity is regulated by the pseudokinase, STE20-related adaptor α (STRADα). The STRADα-LKB1 pathway plays critical roles in epithelial cell polarity, neuronal polarity, and cancer metastasis. Though much attention is given to the STRADα-LKB1 pathway, the function of STRADα itself, including a role outside of the LKB1 pathway, has not been well-studied. Data in Caenorhabditis elegans suggest that STRADα has an LKB1-independent role in regulating cell polarity, and therefore we tested the hypothesis that STRADα regulates cancer cell polarity and motility when wild-type LKB1 is absent. These results show that STRADα protein is reduced in LKB1-null cell lines (mutation or homozygous deletion) and this partial degradation occurs through the Hsp90-dependent proteasome pathway. The remaining STRADα participates in cell polarity and invasion, such that STRADα depletion results in misaligned lamellipodia, improper Golgi positioning, and reduced invasion. To probe the molecular basis of this defect, we show that STRADα associates in a complex with PAK1, and STRADα loss disrupts PAK1 activity via Thr(423) PAK1 phosphorylation. When STRADα is depleted, PAK1-induced invasion could not occur, suggesting that STRADα is necessary for PAK1 to drive motility. Furthermore, STRADα overexpression caused increased activity of the PAK1-activating protein, rac1, and a constitutively active rac1 mutant (Q61L) rescued pPAK(Thr423) and STRADα invasion defects. Taken together, these results show that a STRADα-rac1-PAK1 pathway regulates cell polarity and invasion in LKB1-null cells. It also suggests that while the function of LKB1 and STRADα undoubtedly overlap, they may also have mutually exclusive roles.

Localization-specific LKB1 loss in head and neck squamous cell carcinoma metastasis.

BACKGROUND: LKB1 loss is associated with invasive carcinoma and metastasis. In head and neck squamous cell carcinoma (HNSCC), lymph node metastasis status is the strongest predictor of survival. METHODS: LKB1 immunohistochemistry was performed on primary tumors from patients with HNSCC who never developed metastases, patients with metastases, and their paired lymph node metastasis to determine whether LKB1 was associated with HNSCC metastasis. RESULTS: LKB1 was expressed in the nucleus and cytoplasm of tumor cells and HNSCC cell lines. Primary tumors from patients with metastatic carcinoma had decreased nuclear, but not cytoplasmic, LKB1 compared to patients with nonmetastatic cancer. Nuclear LKB1 levels correlated with lymph node metastasis grade and primary tumor site. The percentage of tumor cells negative for nuclear LKB1 staining was greatest in the primary metastatic tumor group, and decreased nuclear LKB1 trended toward worse disease-free survival. CONCLUSION: Decreased nuclear LKB1 levels correlate with HNSCC metastasis and suggest a nuclear function of LKB1 in repressing HNSCC metastasis.

Long-term exposure to AZT, but not d4T, increases endothelial cell oxidative stress and mitochondrial dysfunction.

Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known about hepatic NRTI toxicity, the mechanism of toxicity in endothelial cells is incompletely understood. Human aortic endothelial and HepG2 liver cells were exposed to 1 muM AZT or d4T for up to 5 weeks. Markers of oxidative stress, mitochondrial function, NRTI phosphorylation, mitochondrial DNA (mtDNA) levels, and cytotoxicity were monitored over time. In endothelial cells, AZT significantly oxidized glutathione redox potential, increased total cellular and mitochondrial-specific superoxide, decreased mitochondrial membrane potential, increased lactate release, and caused cell death from weeks 3 through 5. Toxicity occurred in the absence of di- and tri-phosphorylated AZT and mtDNA depletion. These data show that oxidative stress and mitochondrial dysfunction in endothelial cells occur with a physiologically relevant concentration of AZT, and require long-term exposure to develop. In contrast, d4T did not induce endothelial oxidative stress, mitochondrial dysfunction, or cytotoxicity despite the presence of d4T-triphosphate. Both drugs depleted mtDNA in HepG2 cells without causing cell death. Endothelial cells are more susceptible to AZT-induced toxicity than HepG2 cells, and AZT caused greater endothelial dysfunction than d4T because of its pro-oxidative effects.

The roles of HIV-1 proteins and antiretroviral drug therapy in HIV-1-associated endothelial dysfunction.

Since the emergence of highly active antiretroviral therapy (HAART), human immunodeficiency virus-1 (HIV-1)-infected patients have demonstrated dramatic decreases in viral burden and opportunistic infections, and an overall increase in life expectancy. Despite these positive HAART-associated outcomes, it has become increasingly clear that HIV-1 patients have an enhanced risk of developing cardiovascular disease over time. Clinical studies are instrumental in our understanding of vascular dysfunction in the context of HIV-1 infection. However, most clinical studies often do not distinguish whether HIV-1 proteins, HAART, or a combination of these 2 factors cause cardiovascular complications. This review seeks to address the roles of both HIV-1 proteins and antiretroviral drugs in the development of endothelial dysfunction because endothelial dysfunction is the hallmark initial step of many cardiovascular diseases. We analyze recent in vitro and in vivo studies examining endothelial toxicity in response to HIV-1 proteins or in response to the various classes of antiretroviral drugs. Furthermore, we discuss the multiple mechanisms by which HIV-1 proteins and HAART injure the vascular endothelium in HIV-1 patients. By understanding the molecular mechanisms of HIV-1 protein- and antiretroviral-induced cardiovascular disease, we may ultimately improve the quality of life of HIV-1 patients through better drug design and the discovery of new pharmacological targets.

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration.

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.

Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function.

We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1alpha. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1alpha protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1alpha transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1alpha protein in the human ovarian cancer cell line 1A9 and its beta-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1alpha protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1alpha in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1alpha nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link beta-tubulin drug binding with HIF-1alpha protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients.

Sca-1 negatively regulates proliferation and differentiation of muscle cells.

Satellite cells are tissue-specific stem cells critical for skeletal muscle growth and regeneration. Upon exposure to appropriate stimuli, satellite cells produce progeny myoblasts. Heterogeneity within a population of myoblasts ensures that a subset of myoblasts readily differentiate to form myotubes, whereas other myoblasts remain undifferentiated and thus available for future muscle growth. The mechanisms that contribute to this heterogeneity in myoblasts are largely unknown. We show that satellite cells are Sca-1(neg) but give rise to myoblasts that are heterogeneous for sca-1 expression. The majority of myoblasts are sca-1(neg), rapidly divide, and are capable of undergoing myogenic differentiation to form myotubes. In contrast, a minority population is sca-1(pos), divides slower, and does not readily form myotubes. Sca-1 expression is not static but rather dynamically modulated by the microenvironment. Gain-of-function and loss-of-function experiments demonstrate that sca-1 has a functional role in regulating proliferation and differentiation of myoblasts. Myofiber size of sca-1 null muscles is altered in an age-dependent manner, with increased size observed in younger mice and decreased size in older mice. These studies reveal a novel system that reversibly modulates the myogenic behavior of myoblasts. These studies provide evidence that, rather than being a fixed property, myoblast heterogeneity can be modulated by the microenvironment.