Anticancer innovative therapy: Highlights from the ninth annual meeting.

The Ninth Annual Conference of "Anticancer Innovative Therapy", organized by Fondazione IRCCS Istituto Nazionale dei Tumori di Milano (Fondazione IRCCS INT) and hosted by Hotel Michelangelo, was held in Milan on 25 January 2019. Cutting-edge science was presented in two main scientific sessions: i) pre-clinical evidences and new targets, and ii) clinical translation. The Keynote lecture entitled "Cancer stem cells (CSCs): metabolic strategies for their identification and eradication" presented by M. Lisanti, was one of the highlights of the conference. One key concept of the meeting was how the continuous advances in our knowledge about molecular mechanisms in various fields of research (cancer metabolism reprogramming, epigenetic regulation, transformation/invasiveness, and immunology, among others) are driving cancer research towards more effective personalized antineoplastic strategies. Specifically, recent preclinical data on the following topics were discussed: 1. Polycomb group proteins in cancer; 2. A d16HER2 splice variant is a flag of HER2 addiction across HER2-positive cancers; 3. Studying chromatin as a nexus between translational and basic research; 4. Metabolomic analysis in cancer patients; 5. CDK4-6 cyclin inhibitors: clinical activity and future perspectives as immunotherapy adjuvant; and 6. Cancer stem cells (CSCs): metabolic strategies for their identification and eradication. In terms of clinical translation, several novel approaches were presented: 1. Developing CAR-T cell therapies: an update of preclinical and clinical development at University of North Carolina; 2. Vγ9Vδ2 T-cell activation and immune suppression in multiple myeloma; 3. Predictive biomarkers for real-world immunotherapy: the cancer immunogram model in the clinical arena; and 4. Mechanisms of resistance to immune checkpoint blockade in solid tumors. Overall, the pre-clinical and clinical findings presented could pave the way to identify novel actionable therapeutic targets to significantly enhance the care of persons with cancer.

ESPEN expert group recommendations for action against cancer-related malnutrition.

Patients with cancer are at particularly high risk for malnutrition because both the disease and its treatments threaten their nutritional status. Yet cancer-related nutritional risk is sometimes overlooked or under-treated by clinicians, patients, and their families. The European Society for Clinical Nutrition and Metabolism (ESPEN) recently published evidence-based guidelines for nutritional care in patients with cancer. In further support of these guidelines, an ESPEN oncology expert group met for a Cancer and Nutrition Workshop in Berlin on October 24 and 25, 2016. The group examined the causes and consequences of cancer-related malnutrition, reviewed treatment approaches currently available, and built the rationale and impetus for clinicians involved with care of patients with cancer to take actions that facilitate nutrition support in practice. The content of this position paper is based on presentations and discussions at the Berlin meeting. The expert group emphasized 3 key steps to update nutritional care for people with cancer: (1) screen all patients with cancer for nutritional risk early in the course of their care, regardless of body mass index and weight history; (2) expand nutrition-related assessment practices to include measures of anorexia, body composition, inflammatory biomarkers, resting energy expenditure, and physical function; (3) use multimodal nutritional interventions with individualized plans, including care focused on increasing nutritional intake, lessening inflammation and hypermetabolic stress, and increasing physical activity.

Sex differences in expression and subcellular localization of heart rhythm determinant proteins.

To evaluate sex differences in protein expression in the heart, we performed Western blot studies on a subset of Heart Rhythm Determinant (HRD) proteins. We examined key components of a variety of types of mechanical and electrical junctions including, connexin43, plakophilin-2, N-cadherin and plakoglobin, ankyrin-2 and actin. We describe novel findings in sex differences in cardiac protein expression and membrane localization. For most proteins examined, sex differences were significantly more pronounced in the membrane compartment than in overall expression. These studies extend our previous findings in microarray studies to demonstrate that sex differences in gene expression are likely to confer distinct functional properties on male and female myocardium.

Postobstructive regeneration of kidney is derailed when surge in renal stem cells during course of unilateral ureteral obstruction is halted.

Unilateral ureteral obstruction (UUO), a model of tubulointerstitial scarring (TIS), has a propensity toward regeneration of renal parenchyma after release of obstruction (RUUO). No information exists on the contribution of stem cells to this process. We performed UUO in FVB/N mice, reversed it after 10 days, and examined kidneys 3 wk after RUUO. UUO resulted in attenuation of renal parenchyma. FACS analysis of endothelial progenitor (EPC), mesenchymal stem (MSC) and hematopoietic stem (HSC) cells obtained from UUO kidneys by collagenase-dispersed single-cell suspension showed significant increase in EPC, MSC, and HSC compared with control. After RUUO cortical parenchyma was nearly restored, and TIS score improved by 3 wk. This reversal process was associated with return of stem cells toward baseline level. When animals were chronically treated with nitric oxide synthase (NOS) inhibitor at a dose that did not induce hypertension but resulted in endothelial dysfunction, TIS scores were not different from control UUO, but EPC number in the kidney decreased significantly; however, parenchymal regeneration in these mice was similar to control. Blockade of CXCR4-mediated engraftment resulted in dramatic worsening of UUO and RUUO. Similar results were obtained in caveolin-1-deficient but not -overexpressing mice, reflecting the fact that activation of CXCR4 occurs in caveolae. The present data show increase in EPC, HSC, and MSC population during UUO and a tendency for these cells to decrease to control level during RUUO. These processes are minimally affected by chronic NOS inhibition. Blockade of CXCR4-stromal cell-derived factor-1 (SDF-1) interaction by AMD3100 or caveolin-1 deficiency significantly reduced the UUO-associated surge in stem cells and prevented parenchymal regeneration after RUUO. We conclude that the surge in stem cell accumulation during UUO is a prerequisite for regeneration of renal parenchyma.

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies.

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2alpha-, 1beta-, 2gamma-, 1delta-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in alpha1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with alpha1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, alpha1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells. In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.

Caveolinopathies: mutations in caveolin-3 cause four distinct autosomal dominant muscle diseases.

The caveolin-3 protein is expressed exclusively in muscle cells. Caveolin-3 expression is sufficient to form caveolae-sarcolemmal invaginations that are 50 to 100 nm in diameter. Monomers of caveolin-3 oligomerize to form high molecular mass scaffolding on the cytoplasmic surface of the sarcolemmal membrane. A mutation in one caveolin-3 allele produces an aberrant protein product capable of sequestering the normal caveolin-3 protein in the Golgi apparatus of skeletal muscle cells. Improper caveolin-3 oligomerization and membrane localization result in skeletal muscle T-tubule system derangement, sarcolemmal membrane alterations, and large subsarcolemmal vesicle formation. To date, there have been eight autosomal dominant caveolin-3 mutations identified in the human population. Caveolin-3 mutations can result in four distinct, sometimes overlapping, muscle disease phenotypes: limb girdle muscular dystrophy, rippling muscle disease, distal myopathy, and hyperCKemia. Thus, the caveolin-3 mutant genotype-to-phenotype relation represents a clear example of how genetic background can influence phenotypic outcome. This review examines in detail the reported cases of patients with caveolin-3 mutations and their corresponding muscle disease phenotypes.

Familial isolated hyperCKaemia associated with a new mutation in the caveolin-3 (CAV-3) gene.

An 18 year old man and his mother both presented with persistent, isolated raised serum creatine kinase (hyperCKaemia) without muscle symptoms. Analysis of caveolin-3 protein expression in muscle biopsy of the propositus showed a reduction in the protein. Genetic analysis revealed a new heterozygous mutation in the caveolin-3 (CAV-3) gene: a C-->T transition at nucleotide position 83 in exon 1 leading to a substitution of a proline for a leucine at amino acid position 28 (P28L). This is the first pathogenic mutation in the CAV-3 gene associated with isolated familial hyperCKaemia. It expands the genetic heterogeneity in patients with caveolin-3 deficiency and confirms that caveolin-3 deficiency should be considered in the differential diagnosis of isolated hyperCKaemia.

Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time- and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.

Tyrosine phosphorylation of beta-dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins.

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. However, it remains unknown whether tyrosine-phosphorylated beta-dystroglycan interacts with SH2 domain containing proteins. Here, we show that the tyrosine phosphorylation of beta-dystroglycan is constitutively elevated in v-Src transformed cells. We next reconstituted this phosphorylation event in vivo by transiently coexpressing wild-type c-Src with a fusion protein containing full-length beta-dystroglycan. Our results demonstrate that Src-induced tyrosine phosphorylation of beta-dystroglycan is strictly dependent on the presence of a PPxY motif at its extreme C-terminus. In the nonphosphorylated state, this PPxY motif is normally recognized as a ligand by the WW domain; phosphorylation at this site blocks the binding of certain WW domain containing proteins. Using a GST fusion protein carrying the cytoplasmic tail of beta-dystroglycan, we identified five SH2 domain containing proteins that interact with beta-dystroglycan in a phosphorylation-dependent manner, including c-Src, Fyn, Csk, NCK, and SHC. We localized this binding activity to the PPxY motif by employing a panel of beta-dystroglycan-derived phosphopeptides. In addition, tyrosine phosphorylation of beta-dystroglycan in vivo resulted in the coimmunoprecipitation of the same SH2 domain containing proteins, and this binding event required the beta-dystroglycan C-terminal PPxY motif. We discuss the possibility that tyrosine phosphorylation of the PPxY motif within beta-dystroglycan may act as a regulatory switch to inhibit the binding of certain WW domain containing proteins, while recruiting SH2 domain containing proteins.

Caveolae and caveolin-3 in muscular dystrophy.

Caveolae are vesicular invaginations of the plasma membrane, and function as 'message centers' for regulating signal transduction events. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolar membrane domains in skeletal muscle and in the heart. Several mutations within the coding sequence of the human caveolin-3 gene (located at 3p25) have been identified. Mutations that lead to a loss of approximately 95% of caveolin-3 protein expression are responsible for a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans. By contrast, upregulation of the caveolin-3 protein is associated with Duchenne muscular dystrophy (DMD). Thus, tight regulation of caveolin-3 appears essential for maintaining normal muscle health and homeostasis.

Prolactin negatively regulates caveolin-1 gene expression in the mammary gland during lactation, via a Ras-dependent mechanism.

Caveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development. We show that caveolin-1 expression is significantly down-regulated during late pregnancy and lactation. Upon weaning, mammary gland expression of caveolin-1 rapidly returns to non-pregnant "steady-state" levels. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Virtually identical results were obtained with human mammary epithelial cells (hTERT-HME1) in culture. In addition, we demonstrate that prolactin-mediated down-regulation of caveolin-1 expression occurs at the level of transcriptional control and via a Ras-dependent mechanism. Interestingly, in the mammary gland, both mammary epithelial cells and the surrounding mammary adipocytes show prolactin-mediated down-regulation of caveolin-1. This hormone-dependent regulation of caveolin-1 expression is specific to the mammary fat pad. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of the promoter activity and the synthesis of beta-casein, an established reporter of lactogenic differentiation and milk production. These findings may explain why caveolin-1 levels are normally down-regulated during lactation. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, i.e. lactation, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis.

Adenovirus-mediated expression of caveolin-1 in mouse liver increases plasma high-density lipoprotein levels.

Caveolae are 50-100 nm plasma membrane invaginations, which function in cell signaling and transcytosis, as well as in regulating cellular cholesterol homeostasis. These subcompartments of the plasma membrane are characterized by the presence of caveolin proteins. Recent studies have indicated that caveolae may be involved in the regulation of cellular cholesterol efflux to HDL, as well as selective uptake mediated by SR-BI. In the present study, we have determined the effect of caveolin-1 overexpression in mouse liver on plasma lipoprotein metabolism. We evaluated this effect using an adenovirus-mediated gene delivery system. C57BL/6J mice were injected with adenoviruses encoding either caveolin-1 (Adcav-1) or green fluorescent protein (AdGFP) together with a transactivator adenovirus (AdtTA). We found that, after adenovirus injection, caveolin-1 was overexpressed in hepatocytes. Moreover, the recombinant protein was localized to the plasma membrane. We also found that caveolin-1 overexpression induced a marked change in the lipoprotein profile of injected animals. In caveolin-1 overexpressing animals, plasma HDL-cholesterol levels were found to be approximately 2-fold elevated, as compared with control animals. To determine the effect of caveolin-1 on SR-BI-mediated selective uptake, we infected murine hepatocytes in culture with an adenoviral vector carrying the caveolin-1 cDNA or GFP as a control protein. We show that, in primary cultures of hepatocytes, caveolin-1 inhibits DiI-HDL uptake mediated by SR-BI. This result would mechanistically explain the increased plasma HDL-cholesterol levels we observed in caveolin-1 adenovirus-injected animals. In addition, caveolin-1 expression increased the secretion of apolipoprotein A-I in cultured hepatocytes and increased apolipoprotein A-I plasma levels in mice. Our study therefore demonstrates an important role for caveolin-1 in regulating HDL metabolism.

Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A.

Numerous components of the cAMP-based signaling cascade, namely G-proteins and G- protein coupled receptors, adenylyl cyclase, and protein kinase A (PKA) have been localized to caveolae and shown to be regulated by the caveolar marker proteins, the caveolins. In order to gain mechanistic insights into these processes in vivo, we have assessed the functional interaction of caveolin-1 (Cav-1) with PKA using mutational analysis. As two regions of Cav-1 had previously been implicated in PKA signaling in vitro, we constructed Cav-1 molecules with mutations/deletions in one or both of these domains. Examination of these mutants shows that Cav-1 requires the presence of either the scaffolding domain or the COOH-terminal domain (but not both) to functionally interact with and inhibit PKA. Interestingly, in contrast to the wild-type protein, these Cav-1 mutants are not localized to caveolae microdomains. However, upon coexpression with wild-type Cav-1, a substantial amount of the mutants was recruited to the caveolae membrane fraction. Using the Cav-1 double mutant with both disrupted scaffolding and COOH-terminal domains, we show that wild-type Cav-1's inhibition of PKA signaling can be partially abrogated in a dose-responsive manner; i.e., the mutant acts in a dominant-negative fashion. Thus, this dominant-negative caveolin-1 mutant will be extremely valuable for assessing the functional role of endogenous caveolin-1 in regulating a variety of other signaling cascades.

Ligand-independent activation of oestrogen receptor alpha by caveolin-1.

Expression of caveolin-1 in the human mammary adenocarcinoma cell line MCF-7 causes ligand-independent concentration of oestrogen receptor alpha (ERalpha) in the nucleus, and potentiates ligand-independent and ligand-dependent transcription from an oestrogen response element-driven reporter gene. Furthermore, caveolin-1 co-immunoprecipitates with ERalpha [Schlegel, Wang, Katzenellenbogen, Pestell and Lisanti (1999) J. Biol. Chem. 274, 33551-33556]. In the present study we show that caveolin-1 binds directly to ERalpha. This interaction is mediated by residues 82-101 of caveolin-1 (i.e. the caveolin scaffolding domain) and residues 1-282 of ERalpha. The caveolin-binding domain of ERalpha includes the ligand-independent transactivation domain, activation function (AF)-1, but lacks the hormone-binding domain and the ligand-gated transactivation domain, AF-2. In co-transfection studies, caveolin-1 potentiates the transcriptional activation of ERalpha(1-282), a truncation mutant that has intact AF-1 and DNA-binding domains. Since AF-1 activity is regulated largely by phosphorylation we determined that co-expression with caveolin-1 increased the basal phosphorylation of ERalpha(1-282), but blocked the epidermal growth factor-dependent increase in phosphorylation. Indeed, caveolin-1 interacted with and potentiated the transactivation of an ERalpha mutant that cannot be phosphorylated by extracellular signal-regulated kinase (ERK)1/2 [ERalpha(Ser(118)-->Ala)]. Thus caveolin-1 is a novel ERalpha regulator that drives ERK1/2-independent phosphorylation and activation of AF-1.

Caveolin-1, a putative tumour suppressor gene.

Caveolae ('little caves') are plasma membrane specializations of 50-100 nm in diameter, and the caveolins are structural proteins used by cells to form caveolae. We and other investigators have discovered that caveolae organelles may be important both in normal signal transduction and in the pathogenesis of a number of human diseases, such as cancer. Here we describe the functional roles of the caveolin gene family and summarize the evidence that supports a role for caveolae as mediators of a number of cellular signalling processes, including apoptosis.

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

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.