The Cancer Research UK Stratified Medicine Programme as a model for delivering personalised cancer care.

Genomic screening is routinely used to guide the treatment of cancer patients in many countries. However, several multi-layered factors make this effort difficult to deliver within a clinically relevant timeframe. Here we share the learnings from the CRUK-funded Stratified Medicine Programme for advanced NSCLC patients, which could be useful to better plan future studies.

Identifying and Validating Networks of Oncology Biomarkers Mined From the Scientific Literature.

Biomarkers, as measurements of defined biological characteristics, can play a pivotal role in estimations of disease risk, early detection, differential diagnosis, assessment of disease progression and outcomes prediction. Studies of cancer biomarkers are published daily; some are well characterized, while others are of growing interest. Managing this flow of information is challenging for scientists and clinicians. We sought to develop a novel text-mining method employing biomarker co-occurrence processing applied to a deeply indexed full-text database to generate time-interval-delimited biomarker co-occurrence networks. Biomarkers across 6 cancer sites and a cancer-agnostic network were successfully characterized in terms of their emergence in the published literature and the context in which they are described. Our approach, which enables us to find publications based on biomarker relationships, identified biomarker relationships not known to existing interaction networks. This search method finds relevant literature that could be missed with keyword searches, even if full text is available. It enables users to extract relevant biological information and may provide new biological insights that could not be achieved by individual review of papers.

A Sensitive ALK Immunohistochemistry Companion Diagnostic Test Identifies Patients Eligible for Treatment with Crizotinib.

INTRODUCTION: The availability of high-quality, rigorously validated diagnostic tests that can be broadly implemented is necessary to efficiently identify patients with anaplastic lymphoma kinase (ALK)-positive NSCLC who can potentially benefit from treatment with crizotinib. Here we present data on the recently approved Ventana ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ), the only immunohistochemistry (IHC)-based assay linked to treatment outcome. METHODS: NSCLC specimens prospectively tested for anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by flourescent in situ hybridization (FISH) in the PROFILE 1014 clinical trial of crizotinib versus chemotherapy (N = 1018, including 179 ALK-positive and 754 ALK-negative specimens) were evaluated using the ALK (D5F3) CDx assay. Hazard ratios for progression-free survival comparing crizotinib and chemotherapy for ALK IHC-positive patients and ALK FISH-positive patients, as well as for concordance with the enrollment ALK FISH assay, were determined. RESULTS: Results from both assays were obtained for 933 cases. Percent positive, negative, and overall agreement rates were 86.0% , 96.3%, and 94.3%, respectively. There were 53 discrepant cases, of which 25 were ALK FISH-positive/ALK IHC-negative and 28 were ALK FISH-negative/ALK IHC-positive. The hazard ratios using observed outcomes were 0.401 for ALK FISH-positive/ALK IHC-positive cases and 0.407 for all ALK FISH-positive cases tested with ALK IHC versus 0.454 for all ALK FISH-positive cases enrolled in the trial. Outcome data for ALK FISH-negative/ALK IHC-positive cases were not available for analysis. Between-reader agreement rates for ALK IHC involving three independent laboratories exceeded 98%. CONCLUSIONS: The ALK (D5F3) CDx assay is a stand-alone companion diagnostic test for identification of patients for treatment with crizotinib. This automated assay provides an effective option to accurately and rapidly identify patients with ALK-positive NSCLC. The simple binary scoring algorithm results in high reader-to-reader precision.

Phase I Results from a Study of Crizotinib in Combination with Erlotinib in Patients with Advanced Nonsquamous Non-Small Cell Lung Cancer.

INTRODUCTION: This phase I trial was conducted to determine the safety, maximum tolerated dose (MTD)/recommended phase II dose, and efficacy of crizotinib plus erlotinib in patients with advanced NSCLC. METHODS: Patients with NSCLC and an Eastern Cooperative Oncology Group performance status of 0 to 2 after failure of one or two prior chemotherapy regimens were eligible. Erlotinib, 100 mg, was given continuously once daily starting between day -14 and -7; crizotinib, 200 mg twice daily (dose level 1) or 150 mg twice daily (dose level -1), was added continuously beginning on day 1 of treatment cycle 1. Potential pharmacokinetic interactions between crizotinib and erlotinib were evaluated. RESULTS: Twenty-seven patients received treatment; 26 received crizotinib plus erlotinib. Frequent adverse events were diarrhea, rash, decreased appetite, and fatigue. Dose-limiting toxicities were dehydration, diarrhea, dry eye, dysphagia, dyspepsia, esophagitis and vomiting. The MTD was crizotinib, 150 mg twice daily, with erlotinib, 100 mg once daily. Crizotinib increased the erlotinib area under the concentration-time curve 1.5-fold (dose level -1) and 1.8-fold (dose level 1). The plasma level of crizotinib appeared to be unaffected by coadministration of erlotinib. Two patients whose tumors harbored activating EGFR mutations achieved confirmed partial responses, one at each crizotinib dose level. CONCLUSIONS: The MTD of the combination of crizotinib and erlotinib in patients with advanced NSCLC was crizotinib, 150 mg twice daily, with erlotinib, 100 mg once daily, which is less than the approved dose of either agent. The phase II portion of the study was not initiated.

New methods for ALK status diagnosis in non-small-cell lung cancer: an improved ALK immunohistochemical assay and a new, Brightfield, dual ALK IHC-in situ hybridization assay.

INTRODUCTION: The demonstration of anaplastic lymphoma kinase (ALK) positivity in non-small-cell lung cancer (NSCLC) has been hindered by the technical complexity and interpretative challenges of fluorescence in situ hybridization methods for detection of ALK gene rearrangement and by the inadequate sensitivity of existing immunohistochemistry (IHC) methods for ALK protein detection. In this study, we sought to increase the sensitivity of ALK IHC detection and to develop a brightfield assay for concurrent detection of ALK protein expression and ALK gene rearrangement. METHODS: We developed a horseradish peroxidase-based IHC detection system using the novel, nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) and tyramide. We also developed a dual gene protein ALK assay combining a brightfield break-apart in situ hybridization ALK assay with another sensitive IHC method using the novel, nonendogenous hapten 5-nitro-3-pyrazole. We examined the sensitivity and accuracy of these methods using surgically resected NSCLC cases examined with ALK fluorescence in situ hybridization. RESULTS: The new HQ-tyramide IHC detection system offered readily interpretable staining with substantially greater sensitivity than conventional ALK IHC, and produced heterogeneous and homogeneous patterns of ALK protein staining among ALK-positive NSCLC surgical cases. The new 5-nitro-3-pyrazole-based IHC detection system was similar in ALK detection sensitivity to the HQ-tyramide IHC system and was compatible with the brightfield in situ hybridization assay. CONCLUSION: The new HQ-tyramide IHC reagent system allows more sensitive assessment of ALK protein status in NSCLC cases. The new ALK gene-protein assay allows the concurrent visualization of ALK gene and ALK protein status in single cells, allowing more accurate ALK status determination even in heterogeneous specimens.

Galiximab (anti-CD80)-induced growth inhibition and prolongation of survival in vivo of B-NHL tumor xenografts and potentiation by the combination with fludarabine.

Galiximab is a primatized monoclonal antibody that targets CD80 expressed on malignant B cells and is being studied in the clinic as a potential treatment for follicular NHL. We have recently reported that galiximab signals B-NHL cells in vitro and inhibits cell growth and sensitizes resistant tumor cells to apoptosis by chemotherapeutic drugs. This study was designed to validate the in vitro findings in in vivo in mice. Thus, we examined in vivo the antitumor activity of galiximab used alone and in combination with chemotherapeutic agents in SCID mice bearing human lymphoma xenografts. The in vivo antitumor effects of galiximab used alone and in combination with fludarabine or doxorubicin were determined in solid and disseminated human B-lymphoma tumors grown in SCID mice. Galiximab monotherapy in vivo demonstrated significant antitumor activity in a Raji lymphoma solid tumor model and in an SKW disseminated lymphoma tumor model. There was significant inhibition in tumor growth and prolongation of survival. In vitro, galiximab sensitized Raji cells to apoptosis by both fludarabine and doxorubicin. Tumor growth inhibition was significantly enhanced when the mice were treated with the combination of galiximab and fludarabine. These findings support the potential clinical application of galiximab in combination with chemotherapeutic drugs for the treatment of CD80-expressing hematological malignancies.

Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients following crizotinib treatment.

The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10-11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.

TWEAK signals through JAK-STAT to induce tumor cell apoptosis.

The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK-STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK-STAT dependent, as JAK inhibitors block IFNγ-dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK-STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNß, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNß can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK-STAT pathway to induce IFNß, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.

CD80 (B7.1) is expressed on both malignant B cells and nonmalignant stromal cells in non-Hodgkin lymphoma.

BACKGROUND: CD80 is a member of the B7 family of immune coregulatory proteins that mediate both immune activation and suppression. CD80 in particular has recently been shown to play an important role in supporting immune suppression through interactions with B7-H1. CD80 has been identified as a therapeutic target in non-Hodgkin lymphoma (NHL) based on limited immunohistochemical studies of CD80 expression. Clinical studies have shown that the anti-CD80 antibody galiximab is safe and clinically efficacious in follicular NHL. However, the mechanisms through which targeting CD80 inhibits tumor progression remain poorly understood. METHODS: To further define the potential of CD80 as a therapeutic target in NHL, CD80 expression was evaluated by multicolor flow cytometric analysis of primary lymphoma cell suspensions generated from 241 diagnostic biopsies of patients with NHL. RESULTS: CD80 was expressed on malignant B cells in essentially all cases of follicular lymphoma (97%; n = 115), the majority of cases of diffuse large B-cell lymphoma (90%; n = 69), marginal zone lymphoma (91%; n = 22), mantle cell lymphoma (75%; n = 12), and in about half of small lymphocytic lymphoma cases (43%; n = 23). CD80 was also present on tumor-infiltrating T lymphocytes in nearly all cases. Additionally, CD80 was expressed by non-B, non-T cells in 68 and 44% of cases of follicular and diffuse large B-cell NHL, respectively. CONCLUSIONS: CD80 is expressed on both malignant cells and the nonmalignant cells in NHL. Therapeutic targeting of CD80 will therefore modulate the complex intercellular interactions that define the tumor microenvironment in NHL.

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity.

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

A phase II, single-arm study of the anti-α5ß1 integrin antibody volociximab as monotherapy in patients with platinum-resistant advanced epithelial ovarian or primary peritoneal cancer.

OBJECTIVE: This phase II, multicenter, single-arm, two-stage study in platinum-resistant, advanced epithelial ovarian or primary peritoneal cancer evaluated the efficacy, safety, and tolerability of weekly single-agent volociximab. Pharmacokinetic/pharmacodynamic (PK/PD) studies were also performed. METHODS: Sixteen patients were enrolled in Stage 1. Volociximab was administered at 15mg/kg IV qwk until progression of disease or drug intolerability. Tumor response was assessed every 8weeks. Serum samples for PK or whole blood for the evaluation of circulating tumor cells, endothelial cells, and endothelial progenitor cells were obtained on Days 1, 8, 15, 29, and 50. Ascites from one patient was collected for volociximab concentration analysis. Archived tumor tissue was analyzed by immunohistochemistry (IHC) for α5 integrin expression. RESULTS: Safety data are available on all 16 patients; 14 were evaluable for efficacy. One patient had stable disease at 8weeks. The remaining 13 progressed on treatment. Twelve patients (75%) experienced study-related adverse events (AEs); the most common (≥20%) were headache and fatigue. Three patients experienced possible study-related serious AEs (SAEs): reversible posterior leukoencephalopathy syndrome, pulmonary embolism, and hyponatremia. Peak serum concentrations of volociximab increased 2-3 fold from Day 1 to Day 50. Clinically relevant trough levels were achieved (>150µg/mL). IHC analysis of archived tumor sections showed low-to-moderate expression of α5 integrin on all ovarian cancer tissue evaluated. CONCLUSION: Despite insufficient clinical activity in this refractory patient population to continue the study, weekly volociximab was well tolerated, and the gained understanding of the mechanism of action of volociximab will inform future development efforts.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways.

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Combination of two insulin-like growth factor-I receptor inhibitory antibodies targeting distinct epitopes leads to an enhanced antitumor response.

The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth-inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

Antibody-mediated blockade of integrin alpha v beta 6 inhibits tumor progression in vivo by a transforming growth factor-beta-regulated mechanism.

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

A combinatorial role for NFAT5 in both myoblast migration and differentiation during skeletal muscle myogenesis.

Skeletal muscle regeneration depends on myoblast migration, differentiation and myofiber formation. Isoforms of the nuclear factor of activated T cells (NFAT) family of transcription factors display nonredundant roles in skeletal muscle. NFAT5, a new isoform of NFAT, displays many differences from NFATc1-c4. Here, we examine the role of NFAT5 in myogenesis. NFAT5+/- mice displayed a defect in muscle regeneration with fewer myofibers formed at early times after injury. NFAT5 has a muscle-intrinsic function because inhibition of NFAT5 transcriptional activity caused both a migratory and differentiation defect in cultured myoblasts. We identified Cyr61 as a target of NFAT5 signaling in skeletal muscle cells. Addition of Cyr61 to cells expressing inhibitory forms of NFAT5 rescued the migratory phenotype. These results demonstrate a role for NFAT5 in skeletal muscle cell migration and differentiation. Furthermore, as cell-cell interactions are crucial for myoblast differentiation, these data suggest that myoblast migration and differentiation are coupled and that NFAT5 is a key regulator.

Calcineurin-NFATc signaling pathway regulates AQP2 expression in response to calcium signals and osmotic stress.

The aquaporin (AQP)2 channel mediates the reabsorption of water in renal collecting ducts in response to arginine vasopressin (AVP) and hypertonicity. Here we show that AQP2 expression is induced not only by the tonicity-responsive enhancer binding protein (TonEBP)/nuclear factor of activated T cells (NFAT)5-mediated hypertonic stress response but also by the calcium-dependent calcineurin-NFATc pathway. The induction of AQP2 expression by the calcineurin-NFATc pathway can occur in the absence of TonEBP/NFAT5. Mutational and chromatin immunoprecipitation analyses revealed the existence of functional NFAT binding sites within the proximal AQP2 promoter responsible for regulation of AQP2 by NFATc proteins and TonEBP/NFAT5. Contrary to the notion that TonEBP/NFAT5 is the only Rel/NFAT family member regulated by tonicity, we found that hypertonicity promotes the nuclear translocation of NFATc proteins for the subsequent induction of AQP2 expression. Calcineurin activity was also found to be involved in the induction of TonEBP/NFAT5 expression by hypertonicity, thus further defining the signaling mechanisms that underlie the TonEBP/NFAT5 osmotic stress response pathway. The coordinate regulation of AQP2 expression by both osmotic stress and calcium signaling appears to provide a means to integrate diverse extracellular signals into optimal cellular responses.

Intracellular water homeostasis and the mammalian cellular osmotic stress response.

The cellular response to osmotic stress ensures that the concentration of water inside the cell is maintained within a range that is compatible with biologic function. Single cell organisms are particularly dependent on mechanisms that permit adaptation to osmotic stress because each individual cell is directly exposed to the external environment. Mammals, however, limit osmotic stress by establishing an internal aqueous environment in which intravascular water and electrolytes are subject to sensitive and dynamic, organism-based homeostatic regulation. Recent studies of NFAT5/TonEBP, an essential mammalian osmoregulatory transcription factor, demonstrate the unexpected yet critical significance of cell-based osmotic regulation in vivo. These results highlight the fundamental importance of maintaining intracellular water homeostasis in the face of varying cellular metabolic activity and distinct tissue microenvironments.