Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells.

Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (A-kinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKA-mediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.

Quantitation of human papillomavirus DNA in plasma of oropharyngeal carcinoma patients.

PURPOSE: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. METHODS AND MATERIALS: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16(INK4a) staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. RESULTS: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using (18)F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. CONCLUSIONS: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

A novel aldehyde dehydrogenase-3 activator leads to adult salivary stem cell enrichment in vivo.

PURPOSE: To assess aldehyde dehydrogenase (ALDH) expression in adult human and murine submandibular gland (SMG) stem cells and to determine the effect of ALDH3 activation in SMG stem cell enrichment. EXPERIMENTAL DESIGN: Adult human and murine SMG stem cells were selected by cell surface markers (CD34 for human and c-Kit for mouse) and characterized for various other stem cell surface markers by flow cytometry and ALDH isozymes expression by quantitative reverse transcriptase PCR. Sphere formation and bromodeoxyuridine (BrdUrd) incorporation assays were used on selected cells to confirm their renewal capacity and three-dimensional (3D) collagen matrix culture was applied to observe differentiation. To determine whether ALDH3 activation would increase stem cell yield, adult mice were infused with a novel ALDH3 activator (Alda-89) or with vehicle followed by quantification of c-Kit(+)/CD90(+) SMG stem cells and BrdUrd(+) salispheres. RESULTS: More than 99% of CD34(+) huSMG stem cells stained positive for c-Kit, CD90 and 70% colocalized with CD44, Nestin. Similarly, 73.8% c-Kit(+) mSMG stem cells colocalized with Sca-1, whereas 80.7% with CD90. Functionally, these cells formed BrdUrd(+) salispheres, which differentiated into acinar- and ductal-like structures when cultured in 3D collagen. Both adult human and murine SMG stem cells showed higher expression of ALDH3 than in their non-stem cells and 84% of these cells have measurable ALDH1 activity. Alda-89 infusion in adult mice significantly increased c-Kit(+)/CD90(+) SMG population and BrdUrd(+) sphere formation compared with control. CONCLUSION: This is the first study to characterize expression of different ALDH isozymes in SMG stem cells. In vivo activation of ALDH3 can increase SMG stem cell yield, thus providing a novel means for SMG stem cell enrichment for future stem cell therapy.

Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality.

Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.

Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis.

Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T-cell immunity in progression. Comparing Gal-1-deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immunodeficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immunomodulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.

The RGD domain of human osteopontin promotes tumor growth and metastasis through activation of survival pathways.

BACKGROUND: Human osteopontin (OPN), a known tumor associated protein, exists in different isoforms, whose function is unclear. It also possesses a RGD domain, which has been implicated in diverse function. Here, we use genetic approaches to systematically investigate the function of the RGD domain in different OPN isoforms on tumor progression and metastasis for 2 different solid tumor models. METHODOLOGY/PRINCIPAL FINDINGS: Using isoform-specific qRT-PCR, we found that OPN-A and B were the main isoforms overexpressed in evaluated human tumors, which included 4 soft tissue sarcomas, 24 lung and 30 head and neck carcinomas. Overexpression of either OPN-A or B in two different cell types promoted local tumor growth and lung metastasis in SCID mouse xenografts. However, expression of either isoform with the RGD domain either mutated or deleted decreased tumor growth and metastasis, and resulted in increased apoptosis by TUNEL staining. In vitro, whereas mutation of the RGD domain did not affect cell-cell adhesion, soft agar growth or cell migration, it increased apoptosis under hypoxia and serum starvation. This effect could be mitigated when the RGD mutant cells were treated with condition media containing WT OPN. Mechanistically, the RGD region of OPN inhibited apoptosis by inducing NF-kappaB activation and FAK phosphorylation. Inhibition of NF-kappaB (by siRNA to the p65 subunit) or FAK activation (by a inhibitor) significantly increased apoptosis under hypoxia in WT OPN cells, but not in RGD mutant cells. CONCLUSION/SIGNIFICANCE: Unlike prior reports, our data suggest that the RGD domain of both OPN-A and B promote tumor growth and metastasis mainly by protecting cells against apoptosis under stressed conditions and not via migration or invasion. Future inhibitors directed against OPN should target multiple isoforms and should inhibit cell survival mechanisms that involve the RGD domain, FAK phosphorylation and NF-kappaB activation.

Imaging the unfolded protein response in primary tumors reveals microenvironments with metabolic variations that predict tumor growth.

Cancer cells exist in harsh microenvironments that are governed by various factors, including hypoxia and nutrient deprivation. These microenvironmental stressors activate signaling pathways that affect cancer cell survival. While others have previously measured microenvironmental stressors in tumors, it remains difficult to detect the real-time activation of these downstream signaling pathways in primary tumors. In this study, we developed transgenic mice expressing an X-box binding protein 1 (XBP1)-luciferase construct that served as a reporter for endoplasmic reticulum (ER) stress and as a downstream response for the tumor microenvironment. Primary mammary tumors arising in these mice exhibited luciferase activity in vivo. Multiple tumors arising in the same mouse had distinct XBP1-luciferase signatures, reflecting either higher or lower levels of ER stress. Furthermore, variations in ER stress reflected metabolic and hypoxic differences between tumors. Finally, XBP1-luciferase activity correlated with tumor growth rates. Visualizing distinct signaling pathways in primary tumors reveals unique tumor microenvironments with distinct metabolic signatures that can predict for tumor growth.

The role of Hsp70 and Hsp90 in TGF-beta-induced epithelial-to-mesenchymal transition in rat lens epithelial explants.

PURPOSE: This study investigates the effects of heat shock treatment and the role of Hsp70 and Hsp90 on tranforming growth factor beta 2 (TGF-beta2)-induced epithelial-to-mesenchymal transition (EMT) in rat lens epithelial explants. METHODS: Rat lens epithelial explants from 7 to 10 day-old Wistar rats were dissected and incubated for 24 h before treatment. The explants were divided into eight treatment groups: control (culture medium), fibroblast growth factor-2 (FGF-2), TGF-beta2, and TGF-beta2+FGF-2 under normal culture conditions and heat shocked conditions. The explants were heat shocked at 45 degrees C before treatment with the respective media. H&E staining was performed on whole-mount epithelial explants from each group. Immunofluorescence staining for alpha-smooth muscle actin (alpha-SMA), F-actin, and E-cadherin was also used to determine EMT and fibrotic plaque formation in the lens epithelial explants. Apoptotic cell death was determined using the TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) assay. Confocal microscopy was used to visualize immunoreactivity in the whole-mount epithelial explants. Western blot analysis of alpha-SMA, E-cadherin, Hsp70, and Hsp90 were also performed. RESULTS: TGF-beta2-induced EMT and plaque formation in the lens epithelial explants. The simultaneous treatment of epithelial explants with TGF-beta2+FGF-2-induced the most significant morphological changes and EMT. Heat shock treatment of lens epithelial explants before TGF-beta2 treatment did not inhibit plaque formation, but there was significant reduction of alpha-SMA expression and greater E-cadherin expression when compared to the non-heat shocked TGF-beta2-treated explants. Interestingly, TGF-beta-induced apoptotic cell death was significantly lower in the heat shocked explants compared to the non-heat shock lens explants. Heat-induced accumulation of Hsp70 and Hsp90 expression was reduced in the heat shocked groups at day 4 of treatment. CONCLUSIONS: TGF-beta2-induced EMT was significantly reduced in the heat shocked TGF-beta2 lens epithelial explants. After four days of culture, there is a reduction in expression of Hsp70 and Hsp90 in the heat-shocked groups, indicating that the lens epithelial cells are under a less stressful condition than the non-heat shocked groups. In conclusion, molecular chaperones can play a protective role against TGF-beta2-induced EMT and enhance cell survival.

Lens-specific expression of TGF-beta induces anterior subcapsular cataract formation in the absence of Smad3.

PURPOSE: Smad3, a mediator of TGF-beta signaling has been shown to be involved in the epithelial-to-mesenchymal transformation (EMT) of lens epithelial cells in a lens injury model. In this study, the role of Smad3 in anterior subcapsular cataract (ASC) formation was investigated in a transgenic TGF-beta/Smad3 knockout mouse model. METHODS: TGF-beta1 transgenic mice (containing a human TGF-beta1 cDNA construct expressed under the alphaA-crystallin promoter) were bred with mice on a Smad3-null background to generate mice with the following genotypes: TGF-beta1/Smad3(-/-) (null), TGF-beta1/Smad3(+/-), TGF-beta1/Smad3(+/+), and nontransgenic/Smad3(+/+). Lenses from mice of each genotype were dissected and prepared for histologic or optical analyses. RESULTS: All transgenic TGF-beta1 lenses demonstrated subcapsular plaque formation and EMT as indicated by the expression of alpha-smooth muscle actin. However, the sizes of the plaques were reduced in the TGF-beta1/Smad3(-/-) lenses, as was the level of type IV collagen deposition when compared with TGF-beta1/Smad3(+/-) and TGF-beta1/Smad3(+/+) lenses. An increased number of apoptotic figures was also observed in the plaques of the TGF-beta1/Smad3(-/-) lenses compared with TGF-beta1/Smad3(+/+) littermates. CONCLUSIONS: Lens-specific expression of TGF-beta1 induced ASC formation in the absence of the Smad3 signaling mediator, suggests that alternative TGF-beta-signaling pathways participate in this ocular fibrotic model.

Matrix metalloproteinase inhibitors suppress transforming growth factor-beta-induced subcapsular cataract formation.

The pleotropic morphogen transforming growth factor-beta (TGFbeta) plays an important role in the development of fibrotic pathologies, including anterior subcapsular cataracts (ASCs). ASC formation involves increased proliferation and transition of lens epithelial cells into myofibroblasts, through epithelial-mesenchymal transformation that results in opaque plaques beneath the lens capsule. In this study, we used a previously established TGFbeta-induced rat cataract model to explore the role of matrix metalloproteinases (MMPs) in ASC formation. Treatment of excised rat lenses with TGFbeta resulted in enhanced secretion of MMP-2 and MMP-9. Importantly, co-treatment with two different MMP inhibitors (MMPIs), the broad spectrum inhibitor GM6001 and an MMP-2/9-specific inhibitor, suppressed TGFbeta-induced ASC changes, including the epithelial-mesenchymal transformation of lens epithelial cells. Using an anti-E-cadherin antibody, we revealed that conditioned media from lenses treated with TGFbeta contained a 72-kd E-cadherin fragment, indicative of E-cadherin shedding. This was accompanied by attenuated levels of E-cadherin mRNA. Conditioned media from lenses co-treated with TGFbeta and MMPIs exhibited attenuated levels of the E-cadherin fragment compared with those from TGFbeta-treated lenses. Together, these findings demonstrate that TGFbeta-induced E-cadherin shedding in the lens is mediated by MMPs and that suppression of this phenomenon might explain the mechanism by which MMPIs inhibit ASC plaque formation.

The lens of the eye as a focusing device and its response to stress.

The continued peripheral growth of the lens, resulting in the concentration of older tissue toward the center, has the important optical consequence of producing a lens of variable refractive index. An approach consisting of the projection of fine laser beams through excised lenses in physiological solution has been used for in vitro study of lens optical quality. By varying the separation of the incident beams and/or the wavelength characteristics of the laser used, lens refractive properties and relative transparency may be examined. In the review provided, these optical properties are correlated to lens suture anatomy, lens mitochondrial morphology and function and the function of lens heat shock proteins. In addition, lens spherical aberration is evaluated as a function of accommodation. This work can be highlighted as follows: Mammalian lens suture morphology has a direct impact on lens optical function and, while suture structure of mammalian and avian lenses are very different, they both show an age-related deterioration in morphology and focusing ability. The distribution and appearance of mitochondria of the lens epithelium and superficial fiber cells are similar in all vertebrates. Lens mitochondrial integrity is correlated to lens focusing ability, suggesting a correlation between lens optical properties and lens metabolic function. The induction of cold cataract measured optically in cultured mammalian lenses is enhanced by thermal (heat) shock and this effect is prevented by inhibiting heat shock protein production. Finally, lens accommodative function can be studied by measuring lens refractive change using a physiological model involving an intact accommodative apparatus.

Laser scanning analysis of cold cataract in young and old bovine lenses.

PURPOSE: This research involves the use of a laser scanning instrument to evaluate the formation of cold cataracts in young and older bovine lenses. METHODS: Bovine lenses from 18 (n=14) week old calf eyes and approximately 10 (n=7) year old animals were extracted in a sterile environment. The lenses were placed in a specialized glass chamber with temperature controlled circulating culture medium. The temperature cycle inside the chamber started at 37 degrees C, slowly cooled to 4 degrees C, and then warmed back up to 37 degrees C. A laser scanning system (ScanTox) was used to analyze the optical quality of bovine lenses during a cooling and warming cycle. RESULTS: The relative light transmittance was measured as a function of pixel excitation caused by refracted beams and compared to pre-treatment measures. Each lens from each age group showed a significant decrease in transmittance at 4 degrees C, which recovered when the lenses were warmed to 37 degrees C. Lenses from young eyes showed less loss of refracted beam intensity than lenses from older eyes (32% versus 34%), although the difference was not significant. CONCLUSIONS: The results of the present study indicate cold cataracts can be induced in both old and young bovine lenses, as shown by using a scanning laser instrument.

Hsp70 in bovine lenses during temperature stress.

PURPOSE: To determine the effects of heat shock treatment on cold cataract formation in bovine lenses. METHODS: A laser scanning system (ScanTox) was used to analyze the optical quality of bovine lenses during a cooling and warming cycle. Cycloheximide, a compound that prevents new protein synthesis was used to inhibit inducible heat shock protein 70 (Hsp70) production during heat stress. Cycloheximide was also used to verify that the induction of Hsp70 takes place in lenses during heat stress. Western blots determined the relative accumulation of Hsp70 in urea soluble lens fractions. Lenses from animals approximately 2 years of age (n=60) were used in this experiment. RESULTS: The decrease in relative light transmittance during cold cataract varies for each group of lenses, with the greatest decrease appearing in the heat shock group in culture medium and the least in the control groups and the heat shock group with cycloheximide. The primary result is that heat shock lenses were most affected by the cold cataract (57% decrease in intensity of refracted beam), and that this effect is prevented by cycloheximide. Western blot results show an increase of Hsp70 with heat shock in the urea soluble lens fractions. CONCLUSIONS: The results show that heat shock treatment increased both light scattering and the presence of Hsp70. Also cycloheximde prevented both the heat shock effect and the expression of Hsp70 in bovine lenses.

Mechanisms of ocular toxicity using the in vitro bovine lens and sodium dodecyl sulfate as a chemical model.

Previous work using the in vitro bovine lens as a model has shown a correlation between toxicity and lens optical function and showed much higher sensitivity in detecting irritancy of several surfactants at much lower concentrations than the Draize score. In the current study, cultured bovine lenses were used to study the effects of the surfactant sodium dodecyl sulfate (SDS) on lens optical properties and mitochondrial integrity. Bovine lenses were exposed to SDS (0.1 to 0.00625%) for 30 min and cultured for 24 h. Compared to controls (n = 17), loss of sharp focus was evident immediately following exposure to 0.1% SDS (n = 14, p < 0.0001). At 24 h loss of sharp focus became evident in all groups. Loss of lens transparency, significant increase in lens wet weight, and axial length were seen 24 h postexposure in lenses treated with 0.1 to 0.025% SDS. Confocal analysis 24 h postexposure showed SDS concentration-dependent decrease in number and length of the mitochondria in lens epithelial and superficial cortical fiber cells. The results of this study show a correlation between lens optical properties and metabolic function and together provide a sensitive in vitro model of ocular chemical toxicity. Results of confocal analysis suggest that the mitochondrial integrity of the superficial cortical fiber cells is most sensitive to damage caused by SDS. The results further suggest that recovery of lens metabolic function is necessary for the recovery of lens optical properties.