Novel Intra-Procedural Distensibility Measurement Accurately Predicts Immediate Outcome of Pneumatic Dilatation for Idiopathic Achalasia.

OBJECTIVES: Often 2-3 graduated pneumatic dilatations (PD) are required to treat achalasia as there is no current intra-procedural predictor of clinical response. Distensibility measurements using functional lumen imaging probe (FLIP) may provide an intra-procedural predictor of outcome. Our aim was to determine the optimal criterion for esophagogastric junction (EGJ) distensibility measurements during PD that predicts immediate clinical response. METHODS: EGJ distensibility was prospectively measured using FLIP immediately pre- and post-PD. The EGJ distensibility index (EGJ-DI) was defined as a ratio of the narrowest cross-sectional area and the corresponding intra-bag pressure at 40 ml distension. Immediate and short-term clinical responses were defined as Eckardt score ≤3 assessed 2 weeks Post-PD and at 3-month follow-up, respectively. RESULTS: In 54 patients, we performed thirty-seven 30 mm; twenty 35 mm and six 40 mm PDs. The short-term response rate to the graded PD was 93% (27/29) in newly diagnosed achalasia; 87% (13/15) and 70% (7/10) in those who had relapsed after previous PD and Heller's Myotomy, respectively. Among those demonstrating an immediate response, EGJ-DI increased by an average of 4.5 mm2/mmHg (95% CI (3.5, 5.5) (P<0.001). Within-subject Δ EGJ-DI was highly predictive of immediate clinical response with AUROC of 0.89 (95% CI [0.80, 0.98], P<0.001). An increment in EGJ-DI of 1.8 mm2/mmHg after a single PD predicts an immediate response with an accuracy of 87%. CONCLUSIONS: FLIP-measured Δ EGJ-DI is a novel intra-procedural tool that accurately predicts immediate clinical response to PD in achalasia. This technique may potentially dictate an immediate mechanism to "step-up" dilator size within a single endoscopy session.

Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers.

There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers (NSCLC) and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) and SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and colorectal cancers.

Pemetrexed-based chemotherapy in patients with advanced, ALK-positive non-small cell lung cancer.

BACKGROUND: Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC) is highly responsive to crizotinib. To determine whether ALK-positive NSCLC is also sensitive to pemetrexed, we retrospectively evaluated progression-free survival (PFS) of ALK-positive versus ALK-negative patients who had been treated with pemetrexed-based chemotherapy for advanced NSCLC. PATIENTS AND METHODS: We identified 121 patients with advanced, ALK-positive NSCLC in the USA, Australia, and Italy. For comparison, we evaluated 266 patients with advanced, ALK-negative, epidermal growth factor receptor (EGFR)-wild-type NSCLC, including 79 with KRAS mutations and 187 with wild-type KRAS (WT/WT/WT). We determined PFS on different pemetrexed regimens. RESULTS: Among 70 ALK-positive patients treated with a platinum/pemetrexed regimen, the median PFS (mPFS) was 7.3 months (95% confidence interval (CI) 5.5-9.5). The mPFS of 51 ALK-positive patients treated with single-agent pemetrexed or nonplatinum/pemetrexed combinations was 5.5 months (2.8-9.0). For ALK-negative patients, PFS on all pemetrexed-based regimens was similar to that of ALK-positive patients, except in the specific setting of first-line platinum/pemetrexed where the mPFS was only 4.2 and 5.4 months in KRAS and WT/WT/WT patients, respectively. However, among patients with a never/light-smoking history (0-10 pack-year smoking history) treated with first-line platinum/pemetrexed, there was no difference in PFS between ALK-positive and ALK-negative patients. CONCLUSIONS: PFS on pemetrexed or nonplatinum/pemetrexed combinations was similar in ALK-positive and ALK-negative patients. PFS on first-line platinum/pemetrexed may be prolonged in never/light-smoking patients regardless of ALK status.

Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice.

BACKGROUND: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming essential to optimal cancer care. METHODS: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping. RESULTS: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ≥1 mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%). Unanticipated mutations were observed at lower frequencies in IDH and ß-catenin. We observed several associations between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers. Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients with advanced disease toward a genotype-directed targeted therapy. CONCLUSIONS: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice.

Prognostic significance of AMP-activated protein kinase expression and modifying effect of MAPK3/1 in colorectal cancer.

BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.

H1047R phosphatidylinositol 3-kinase mutant enhances HER2-mediated transformation by heregulin production and activation of HER3.

Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K, but not E545K PI3K, markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors.

Amplification of EGFR T790M causes resistance to an irreversible EGFR inhibitor.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib are effective therapies against mutant non-small cell lung cancers (NSCLCs). Treatment is limited by the development of resistance in part explained by the gain of a secondary EGFR mutation, T790M, at the gatekeeper residue. Irreversible EGFR inhibitors, including PF00299804, are effective in vitro and in vivo against EGFR mutant tumors that contain EGFR T790M and are currently under clinical development. In this study, we generate models of resistance to PF00299804, using cell lines with EGFR T790M and show that the PF00299804-resistant models develop focal amplification of EGFR that preferentially involves the T790M-containing allele. These PF00299804-resistant cell lines remain dependent on EGFR for growth as downregulation of EGFR by shRNA compromises their viability. We show that resistance to PF00299804 arises, at least in part, through selection of a pre-existing EGFR T790M-amplified clone both in vitro and using a xenograft model in vivo. Our findings show that EGFR T790M is a common resistance mechanism to both reversible, and when amplified, the irreversible EGFR kinase inhibitors further emphasizing the need to develop more potent therapies against EGFR T790M. These findings can be used to guide studies of patient tumor specimens from ongoing clinical trials of irreversible EGFR kinase inhibitors.

Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase: targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (TYR-14).

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.

Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level.

Tumor necrosis factor alpha-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3-L1 adipocytes.

Tumor necrosis factor-alpha (TNFalpha) has been implicated as a contributing mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previous studies have demonstrated that TNFalpha confers insulin resistance by promoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphorylation of IRS-1. However, little is known about which signaling molecules are involved in this process in adipocytes and about the temporal sequence of events that ultimately leads to TNFalpha-stimulated IRS-1 serine phosphorylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathway restore insulin signaling to normal levels despite the presence of TNFalpha. Additional experiments show that MEK1/2 activity is required for TNFalpha-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling. We observe that TNFalpha requires 2.5-4 h to markedly reduce insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNFalpha activates p42/44 MAP kinase, maximal stimulation is observed within 10-30 min. To our surprise, p42/44 activity returns to basal levels well before IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated than a direct phosphorylation of IRS-1 triggered by the early spike of TNFalpha-induced p42/44 activity. Chronic TNFalpha treatment (>> 72 h) causes adipocyte dedifferentiation, as evidenced by the loss of triglycerides and down-regulation of adipocyte-specific markers. We observe that this longer term TNFalpha-mediated dedifferentiation effect utilizes alternative, p42/44 MAP kinase-independent intracellular pathways. This study suggests that TNFalpha-mediated insulin resistance, but not adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathway.

Gene-target recognition among members of the myc superfamily and implications for oncogenesis.

Myc and Mad family proteins regulate multiple biological processes through their capacity to influence gene expression directly. Here we show that the basic regions of Myc and Mad proteins are not functionally equivalent in oncogenesis, have separable E-box-binding activities and engage both common and distinct gene targets. Our data support the view that the opposing biological actions of Myc and Mxi1 extend beyond reciprocal regulation of common gene targets. Identification of differentially regulated gene targets provides a framework for understanding the mechanism through which the Myc superfamily governs the growth, proliferation and survival of normal and neoplastic cells.

Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis.

Although much has been learned regarding the importance of p38 mitogen-activated protein kinase in inflammatory and stress responses, relatively little is known concerning its role in differentiation processes. Recently, we demonstrated that p38 mitogen-activated protein kinase activity is necessary for the differentiation of 3T3-L1 fibroblasts into adipocytes (Engelman, J. A., Lisanti, M. P., and Scherer, P. E. (1998) J. Biol. Chem. 273, 32111-32120). p38 activity is high during the initial stages of differentiation but decreases drastically as the fibroblasts undergo terminal differentiation into adipocytes. However, it remains unknown whether activation of p38 is sufficient to stimulate adipogenesis and whether the down-regulation of p38 activity in mature adipocytes is critical for maintaining adipocyte homeostasis. In this report, we have directly addressed these questions by analyzing 3T3-L1 cell lines harboring a specific upstream activator of p38 (a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) mutant, MKK6(Glu)) under the control of an inducible promoter. Induction of MKK6(Glu) in 3T3-L1 fibroblasts spurs adipocyte conversion in the absence of the hormonal mixture normally required for efficient differentiation of wild-type cells. However, activation of p38 in adipocytes leads to cell death. Furthermore, treatment of 3T3-L1 fibroblasts with salicylate, a potent stimulator of p38, produces adipocyte-specific changes consistent with those observed with induction of MKK6(Glu). Expression of MKK6(Glu) in NIH-3T3 fibroblasts (cells that do not differentiate into adipocytes under normal conditions) is capable of converting these fibroblasts into lipid-laden fat cells following hormonal stimulation. Thus, p38 activation has pro-adipogenic effects in multiple fibroblast cell lines.

p42/44 MAP kinase-dependent and -independent signaling pathways regulate caveolin-1 gene expression. Activation of Ras-MAP kinase and protein kinase a signaling cascades transcriptionally down-regulates caveolin-1 promoter activity.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are down-regulated in NIH 3T3 cells in response to transformation by activated oncogenes, such as H-Ras(G12V) and v-Abl. The mechanisms governing this down-regulation event remain unknown. Here, we show that caveolin-1 gene expression is directly regulated by activation of the Ras-p42/44 MAP kinase cascade. Down regulation of caveolin-1 protein expression by Ras is independent of (i) the type of activating mutation (G12V versus Q61L) and (ii) the form of activated Ras transfected (H-Ras versus K-Ras versus N-Ras). Treatment of Ras or Raf-transformed NIH 3T3 cells with a well characterized MEK inhibitor (PD 98059) restores caveolin-1 protein expression. In contrast, treatment of v-Src and v-Abl transformed NIH 3T3 cells with PD 98059 does not restore caveolin-1 expression. Thus, there must be at least two pathways for down-regulating caveolin-1 expression: one that is p42/44 MAP kinase-dependent and another that is p42/44 MAP kinase-independent. We focused our efforts on the p42/44 MAP kinase-dependent pathway. The activity of a panel of caveolin-1 promoter constructs was evaluated using transient expression in H-Ras(G12V) transformed NIH 3T3 cells. We show that caveolin-1 promoter activity is up-regulated approximately 5-fold by inhibition of the p42/44 MAP kinase cascade. Using electrophoretic mobility shift assays we provide evidence that the caveolin-1 promoter (from -156 to -561) is differentially bound by transcription factors in normal and H-Ras(G12V)-transformed cells. We also show that activation of protein kinase A (PKA) signaling is sufficient to down-regulate caveolin-1 protein expression and promoter activity. Thus, we have identified two signaling pathways (Ras-p42/44 MAP kinase and PKA) that transcriptionally down-regulate caveolin-1 gene expression.

Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation.

Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.

Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. Retention of caveolin-2 at the level of the golgi complex.

Caveolins-1 and -2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and -2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.

Sequence and detailed organization of the human caveolin-1 and -2 genes located near the D7S522 locus (7q31.1). Methylation of a CpG island in the 5' promoter region of the caveolin-1 gene in human breast cancer cell lines.

The CA microsatellite repeat marker, D7S522, is located at the center of a approximately 1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (approximately 100-200 kb) containing D7S522 and the human genes encoding caveolins 1 and 2. Here, we present the detailed organization of the caveolin locus and its relationship to D7S522, as deduced using a shot-gun sequencing approach. We derived two adjacent contigs for a total coverage of approximately 250 kb. Analysis of these contigs reveals that D7S522 is located approximately 67 kb upstream of the caveolin-2 gene and that the caveolin-2 gene is located approximately 19 kb upstream of the caveolin-1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the caveolin genes; these include the intron-exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the caveolin genes. The first and second exons of both caveolin genes are embedded within CpG islands. These results suggest that regulation of caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5' promoter region of the caveolin-1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T-47D) that fail to express the caveolin-1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non-methylated and these cells express high levels of the caveolin-1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the caveolin-1/-2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the caveolin genes in a variety of human tumors.

Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo.

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.