Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis.

The tumor microenvironment plays a critical role in cancer progression, but the precise mechanisms by which stromal cells influence the epithelium are poorly understood. Here we show that p62 levels were reduced in the stroma of several tumors and that its loss in the tumor microenvironment or stromal fibroblasts resulted in increased tumorigenesis of epithelial prostate cancer cells. The mechanism involves the regulation of cellular redox through an mTORC1/c-Myc pathway of stromal glucose and amino acid metabolism, resulting in increased stromal IL-6 production, which is required for tumor promotion in the epithelial compartment. Thus, p62 is an anti-inflammatory tumor suppressor that acts through the modulation of metabolism in the tumor stroma.

Targeting and cytotoxicity of SapC-DOPS nanovesicles in pancreatic cancer.

Only a small number of promising drugs target pancreatic cancer, which is the fourth leading cause of cancer deaths with a 5-year survival of less than 5%. Our goal is to develop a new biotherapeutic agent in which a lysosomal protein (saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS) are assembled into nanovesicles (SapC-DOPS) for treating pancreatic cancer. A distinguishing feature of SapC-DOPS nanovesicles is their high affinity for phosphatidylserine (PS) rich microdomains, which are abnormally exposed on the membrane surface of human pancreatic tumor cells. To evaluate the role of external cell PS, in vitro assays were used to correlate PS exposure and the cytotoxic effect of SapC-DOPS in human tumor and nontumorigenic pancreatic cells. Next, pancreatic tumor xenografts (orthotopic and subcutaneous models) were used for tumor targeting and therapeutic efficacy studies with systemic SapC-DOPS treatment. We observed that the nanovesicles selectively killed human pancreatic cancer cells in vitro by inducing apoptotic death, whereas untransformed cells remained unaffected. This in vitro cytotoxic effect correlated to the surface exposure level of PS on the tumor cells. Using xenografts, animals treated with SapC-DOPS showed clear survival benefits and their tumors shrank or disappeared. Furthermore, using a double-tracking method in live mice, we showed that the nanovesicles were specifically targeted to orthotopically-implanted, bioluminescent pancreatic tumors. These data suggest that the acidic phospholipid PS is a biomarker for pancreatic cancer that can be effectively targeted for therapy utilizing cancer-selective SapC-DOPS nanovesicles. This study provides convincing evidence in support of developing a new therapeutic approach to pancreatic cancer.

c-Myc phosphorylation by PKCζ represses prostate tumorigenesis.

Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.

Determining α-helical and ß-sheet secondary structures via pulsed electron spin resonance spectroscopy.

A new method has been developed to determine α-helical and ß-sheet secondary structural components of aqueous and membrane-bound proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The three-pulse electron spin echo envelope modulation (ESEEM) technique was used to detect weakly coupled (2)H-labeled nuclei on side chains in the proximity of a strategically placed nitroxide spin-label up to 8 Å away. Changes in the ESEEM spectra for different samples correlate directly to periodic structural differences between α-helical and ß-sheet motifs. These distinct trends were demonstrated with α-helical (M2δ subunit of the acetylcholine receptor) and ß-sheet (ubiquitin) peptides in biologically relevant sample environments.

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy.

Wild-type phospholamban (WT-PLB), a Ca(2+)-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca(2+)-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100ns rotational correlation time (τ(c)); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.

Cytotoxicity and Selectivity in Skin Cancer by SapC-DOPS Nanovesicles.

Squamous cell carcinoma (SCC) and melanoma are malignant human cancers of the skin with an annual mortality that exceed 10,000 cases every year in the USA alone. In this study, the lysosomal protein saposin C (SapC) and the phospholipid dioloylphosphatidylserine (DOPS) were assembled into cancer-selective nanovesicles (SapC-DOPS) and successfully tested using several in vitro and in vivo skin cancer models. Using MTT assay that measures the percentage of cell death, SapC-DOPS cytotoxic effect on three skin tumor cell lines (squamous cell carcinoma, SK-MEL-28, and MeWo) was compared to two normal nontumorigenic skin cells lines, normal immortalized keratinocyte (NIK) and human fibroblast cell (HFC). We observed that the nanovesicles selectively killed the skin cancer cells by inducing apoptotic cell death whereas untransformed skin cancer cells remained unaffected. Using subcutaneous skin tumor xenografts, animals treated with SapC-DOPS by subcutaneous injection showed a 79.4 % tumor reduced compared to the control after 4 days of treatment. We observed that the nanovesicles killed skin cancer cells by inducing apoptotic cell death compared to the control as revealed by TUNEL staining of xenograft tumor sections.

p62 is a key regulator of nutrient sensing in the mTORC1 pathway.

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.

(15)N Solid-state NMR spectroscopic studies on phospholamban at its phosphorylated form at ser-16 in aligned phospholipid bilayers.

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically (15)N-Ala-11- and (15)N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned (15)N-labeled Ala-11 and Leu-7 WT-PLB samples show (15)N resonance peaks at approximately 71ppm and 75ppm, respectively, while the corresponding phosphorylated forms P-PLB show (15)N peaks at 92ppm and 99ppm, respectively. These (15)N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.

The Par-4/PTEN connection in tumor suppression.

Tumor suppressors function in a coordinated regulatory network, and their inactivation is a key step in carcinogenesis. The tumor suppressor Par-4 is a novel integral player in the PTEN network. Thus, Par-4 is absent in a high percentage of human prostate carcinomas, and its loss is concomitantly associated with PTEN loss. Genetic ablation of Par-4 induces fully invasive prostate carcinomas in PTEN-heterozygous mice. In contrast, Par-4 deficiency alone, like PTEN heterozygosis, results in lesions that are unable to progress beyond the benign neoplastic stage known as PIN. At this PIN transition, the mutual induction of Par-4 and PTEN is an additional regulatory step in preventing cancer progression. Par-4 deficiency cooperates with PTEN haploinsufficiency in prostate cancer initiation and progression and their simultaneous inactivation, in addition to enhancing Akt activation, sets in motion a unique mechanism involving the synergistic activation of NFkappaB. These results suggest that the concurrent interruption of complementary signaling pathways targeting PI3K/Akt and NFkappaB activation could provide new and effective strategies for cancer therapy.

Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-kappaB activation and invasive prostate carcinoma.

Prostate cancer is one of the most common neoplasias in men. The tumor suppressor Par-4 is an important negative regulator of the canonical NF-kappaB pathway and is highly expressed in prostate. Here we show that Par-4 expression is lost in a high percentage of human prostate carcinomas, and this occurs in association with phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss. Par-4 null mice, similar to PTEN-heterozygous mice, only develop benign prostate lesions, but, importantly, concomitant Par-4 ablation and PTEN-heterozygosity lead to invasive prostate carcinoma in mice. This strong tumorigenic cooperation is anticipated in the preneoplastic prostate epithelium by an additive increase in Akt activation and a synergistic stimulation of NF-kappaB. These results establish the cooperation between Par-4 and PTEN as relevant for the development of prostate cancer and implicate the NF-kappaB pathway as a critical event in prostate tumorigenesis.

Protein kinase Czeta represses the interleukin-6 promoter and impairs tumorigenesis in vivo.

Gene alterations in tumor cells that confer the ability to grow under nutrient- and mitogen-deficient conditions constitute a competitive advantage that leads to more-aggressive forms of cancer. The atypical protein kinase C (PKC) isoform, PKCzeta, has been shown to interact with the signaling adapter p62, which is important for Ras-induced lung carcinogenesis. Here we show that PKCzeta-deficient mice display increased Ras-induced lung carcinogenesis, suggesting a new role for this kinase as a tumor suppressor in vivo. We also show that Ras-transformed PKCzeta-deficient lungs and embryo fibroblasts produced more interleukin-6 (IL-6), which we demonstrate here plays an essential role in the ability of Ras-transformed cells to grow under nutrient-deprived conditions in vitro and in a mouse xenograft system in vivo. We also show that PKCzeta represses histone acetylation at the C/EBPbeta element in the IL-6 promoter. Therefore, PKCzeta, by controlling the production of IL-6, is a critical signaling molecule in tumorigenesis.

The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy.

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.

Side chain and backbone dynamics of phospholamban in phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy.

2H and 15N solid-state NMR spectroscopic techniques were used to investigate both the side chain and backbone dynamics of wild-type phospholamban (WT-PLB) and its phosphorylated form (P-PLB) incorporated into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) phospholipid bilayers. 2H NMR spectra of site-specific CD3-labeled WT-PLB (at Leu51, Ala24, and Ala15) in POPC bilayers were similar under frozen conditions (-25 degrees C). However, significant differences in the line shapes of the 2H NMR spectra were observed in the liquid crystalline phase at and above 0 degrees C. The 2H NMR spectra indicate that Leu51, located toward the lower end of the transmembrane (TM) helix, shows restricted side chain motion, implying that it is embedded inside the POPC lipid bilayer. Additionally, the line shape of the 2H NMR spectrum of CD3-Ala24 reveals more side chain dynamics, indicating that this residue (located in the upper end of the TM helix) has additional backbone and internal side chain motions. 2H NMR spectra of both WT-PLB and P-PLB with CD3-Ala15 exhibit strong isotropic spectral line shapes. The dynamic isotropic nature of the 2H peak can be attributed to side chain and backbone motions to residues located in an aqueous environment outside the membrane. Also, the spectra of 15N-labeled amide WT-PLB at Leu51 and Leu42 residues showed only a single powder pattern component indicating that these two 15N-labeled residues located in the TM helix are motionally restricted at 25 degrees C. Conversely, 15N-labeled amide WT-PLB at Ala11 located in the cytoplasmic domain showed both powder and isotropic components at 25 degrees C. Upon phosphorylation, the mobile component contribution increases at Ala11. The 2H and 15N NMR data indicate significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.

Investigating the interaction of saposin C with POPS and POPC phospholipids: a solid-state NMR spectroscopic study.

The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.

Phospholamban and its phosphorylated form interact differently with lipid bilayers: a 31P, 2H, and 13C solid-state NMR spectroscopic study.

Phospholamban (PLB) is a 52-amino acid integral membrane protein that helps to regulate the flow of Ca(2+) ions in cardiac muscle cells. Recent structural studies on the PLB pentamer and the functionally active monomer (AFA-PLB) debate whether its cytoplasmic domain, in either the phosphorylated or dephosphorylated states, is alpha-helical in structure as well as whether it associates with the lipid head groups (Oxenoid, K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 10870-10875; Karim, C. B. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 14437-14442; Andronesi, C.A. (2005) J. Am. Chem. Soc. 127, 12965-12974; Li, J. (2003) Biochemistry 42, 10674-10682; Metcalfe, E. E. (2005) Biochemistry 44, 4386-4396: Clayton, J. C. (2005) Biochemistry 44, 17016-17026). Comparing the secondary structure of the PLB pentamer and its phosphorylated form (P-PLB) as well as their interaction with the lipid bilayer is crucial in order to understand its regulatory function. Therefore, in this study, the full-length wild-type (WT) PLB and P-PLB were incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) phospholipid bilayers and studied utilizing solid-state NMR spectroscopy. The analysis of the (2)H and (31)P solid-state NMR data of PLB and P-PLB in POPC multilamellar vesicles (MLVs) indicates that a direct interaction takes place between both proteins and the phospholipid head groups. However, the interaction of P-PLB with POPC bilayers was less significant compared that with PLB. Moreover, the secondary structure using (13)C=O site-specific isotopically labeled Ala15-PLB and Ala15-P-PLB in POPC bilayers suggests that this residue, located in the cytoplasmic domain, is a part of an alpha-helical structure for both PLB and P-PLB.

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing (13)C CPMAS, (2)H, and REDOR solid-state NMR spectroscopy.

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca(2+) regulator. (13)C cross-polarization magic angle spinning spectra of (13)C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding (13)C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an alpha-helical conformation as monitored by an enhancement of the alpha-helical carbonyl (13)C resonance in the corresponding NMR spectrum. (13)C-(15)N REDOR solid-state NMR spectroscopic experiments revealed the distance between the (13)C carbonyl carbon of Leu39 and the (15)N amide nitrogen of Leu42 to be 4.2+/-0.2A indicating an alpha-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three (2)H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11 degrees +/-5 degrees tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing 31P and 2H solid-state NMR spectroscopy.

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using (2)H and (31)P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 degrees C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S(CD)) were calculated from the dePaked (2)H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S(CD) profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S(CD) profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S(CD) profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The (31)P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].

Solid-state NMR spectroscopic studies of an integral membrane protein inserted into aligned phospholipid bilayer nanotube arrays.

This communication demonstrates for the first time that solid-state NMR spectroscopic studies can be used to investigate aligned phospholipid bilayer nanotube arrays. Also, an integral membrane peptide can be successfully incorporated into the oriented phospholipid bilayer nanotube arrays and studied with 2H solid-state NMR spectroscopy.