Erik R. Kline

Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor-1α 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.

Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation

Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition and reduced patient survival. As vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data showed that WFA had weak cytotoxic and apoptotic activity at concentrations less than or equal to 500 nM, but retained potent anti‐invasive activity at these low doses. Imaging of breast cancer cell lines revealed that WFA induces perinuclear vimentin accumulation followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin‐binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti‐invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2 μM in plasma with a half‐life of 1.36 hr following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose‐dependent inhibition of metastatic lung nodules and induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti‐metastatic agent and the anti‐metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.

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.

Vimentin regulates lung cancer cell adhesion through a VAV2–Rac1 pathway to control focal adhesion kinase activity

Vimentin is an intermediate filament protein whose expression correlates with increased metastatic disease, reduced patient survival and poor prognosis across multiple tumor types. Despite these well-characterized correlations, the molecular role of vimentin in cancer cell motility remains undefined. To approach this, we used an unbiased phosphoproteomics screen in lung cancer cell lines to discover cell motility proteins that show significant changes in phosphorylation upon vimentin depletion. We identified the guanine nucleotide exchange factor (GEF), VAV2, as having the greatest loss of phosphorylation owing to vimentin depletion. Since VAV2 serves as a GEF for the small Rho GTPase Rac1, a key player in cell motility and adhesion, we explored the vimentin-VAV2 pathway as a potential novel regulator of lung cancer cell motility. We show that VAV2 localizes to vimentin-positive focal adhesions (FAs) in lung cancer cells and complexes with vimentin and FA kinase (FAK). Vimentin loss impairs both pY142-VAV2 and downstream pY397-FAK activity showing that vimentin is critical for maintaining VAV2 and FAK activity. Importantly, vimentin depletion reduces the activity of the VAV2 target, Rac1, and a constitutively active Rac1 rescues defects in FAK and cell adhesion when vimentin or VAV2 is compromised. Based upon this data, we propose a model whereby vimentin promotes FAK stabilization through VAV2-mediated Rac1 activation. This model may explain why vimentin expressing metastatic lung cancer cells are more motile and invasive.

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.

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

Background: LKB1 is a serine/threonine kinase important for cell polarity and motility. Results: LKB1 loss causes focal adhesion kinase hyperactivation and aberrant cell motility. Conclusion: LKB1 represses focal adhesion kinase to regulate its turnover. Significance: This provides information on how LKB1 regulates the cell adhesion pathway during cell motility. Liver kinase β1 (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 Tyr397/Tyr861 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 Tyr397-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

Background: STRADα is the cofactor of the tumor suppressor LKB1; however, it is unclear if STRADα has LKB1-independent roles. Results: STRADα complexes with the kinase PAK1 to modify PAK1 phosphorylation likely via rac1 and control cell motility when LKB1 is null. Conclusion: STRADα regulates PAK1 in LKB1-null cells to oversee cancer cell polarity and invasion. Significance: This shows an undiscovered role of STRADα distinct from the LKB1 pathway. 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 Thr423 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 pPAKThr423 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

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.

Vimentin: A Novel Chemopreventive Target for Breast Cancer Metastasis.

Background: Cancer metastasis is the major cause of death in nearly all tumor types; however, most treatments target the primary tumor and not the highly invasive metastatic cells. We propose to prevent cancer metastasis by precisely targeting the cancer invasion machinery. To do this we disrupt a novel target-the vimentin cytoskeleton. Vimentin is overexpressed in nearly all invasive solid tumors and is critical for cell migration. Here we show that Withania sominifera root extracts (WRE) and one of its primary constituent, Withaferin A (WFA), targets vimentin to inhibit cancer cell motility and migration while having negligible effects on viability. WFA is a steroidal natural product with 4-rings (A-D) connected to a lactone ring. We test the hypothesis that WFA is an anti-invasive compound that disrupts vimentin function with limited toxicity.Material and Methods : We employ cutting-edge live cell confocal imaging studies in combination with traditional molecular biology techniques to dissect the precise mechanism of how WRE or WFA inhibits cancer cell migration and invasion. We have employed a wounding model and Matrigel invasion assay to determine the anti-migratory and anti-invasive properties of WRE and WFA using MDAMB-231 cells. We have used GFP-vimentin to visualize the changes in vimentin dynamics. Effect of WRE and WFA on cell cycle was analyzed by Flow cytometry.Results: Our results show WFA has weak anti-proliferative activity at low concentrations but potently inhibits breast cancer migration and invasion in a dose-dependent manner. High resolution confocal images reveal that in WFA-treated cells, vimentin fails to invade the lamellipodia resulting in decreased cell migration. This is supported by live cell confocal imaging showing that at high doses WFA treatment depolymerizes GFP: vimentin. Furthermore, treatment with WFA results in aberrant vimentin phosphorylation, likely resulting in a net depolymerized phenotype. In order to determine if the vimentin-binding A ring of WFA is critical for its anti-invasive efficacy, we synthesized and tested A-ring modified analogs of WFA. All three analogs showed severely reduced potency in inhibiting cancer cell migration and invasion, suggesting that the A ring is critical for its anti-invasive activity.Discussion: The present study shows that WRE or WFA inhibits breast cancer cell migration at nanomolar concentrations by disrupting vimentin at concentrations that have minimal effect on proliferation. We believe its strong anti-migratory activity make this an appropriate compound as an anti-metastatic chemopreventative. Ultimately, we envision WFA can be used as a vimentin-targeting chemopreventative in high-risk metastatic patients and has the potential to be used with traditional cytotoxics. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5063.

Abstract A11: STRADα regulates cell polarity and invasion through PAK1 signaling in LKB1 null cells

The kinase LKB1 is mutated in multiple tumor types with a high mutation rate in non-small cell lung cancer. LKB1 activity is regulated by the pseudokinase, STE20-related adaptor alpha (STRADα), and the STRADα-LKB1 pathway plays critical roles in cell polarity and 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 C. 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 thr423 PAK1 phosphorylation. When STRADα is depleted, PAK1-induced invasion could not occur, suggesting that STRADα is necessary for PAK1 to drive motility. Based upon these data, we conclude that STRADα regulates PAK1 in LKB1-null cells to oversee cancer cell polarity and invasion.