Targeting Intrinsically Disordered Transcription Factors: Changing the Paradigm.

Increased understanding of intrinsically disordered proteins (IDPs) and protein regions has revolutionized our view of the relationship between protein structure and function. Data now support that IDPs can be functional in the absence of a single, fixed, three-dimensional structure. Due to their dynamic morphology, IDPs have the ability to display a range of kinetics and affinity depending on what the system requires, as well as the potential for large-scale association. Although several studies have shed light on the functional properties of IDPs, the class of intrinsically disordered transcription factors (TFs) is still poorly characterized biophysically due to their combination of ordered and disordered sequences. In addition, TF modulation by small molecules has long been considered a difficult or even impossible task, limiting functional probe development. However, with evolving technology, it is becoming possible to characterize TF structure-function relationships in unprecedented detail and explore avenues not available or not considered in the past. Here we provide an introduction to the biophysical properties of intrinsically disordered TFs and we discuss recent computational and experimental efforts toward understanding the role of intrinsically disordered TFs in biology and disease. We describe a series of successful TF targeting strategies that have overcome the perception of the "undruggability" of TFs, providing new leads on drug development methodologies. Lastly, we discuss future challenges and opportunities to enhance our understanding of the structure-function relationship of intrinsically disordered TFs.

Small molecule inhibitors of ezrin inhibit the invasive phenotype of osteosarcoma cells.

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis.

Neuropeptide Y and its Y2 receptor: potential targets in neuroblastoma therapy.

Neuroblastomas are pediatric tumors that develop from sympathetic precursors and express neuronal proteins, such as neuropeptide Y (NPY). NPY is a sympathetic neurotransmitter acting via multiple receptors (Y1-Y5R). Both NPY and Y2Rs are commonly expressed in neuroblastoma cell lines and tissues. The peptide secreted from neuroblastomas stimulates tumor cell proliferation and angiogenesis. As both processes are Y2R-mediated, the aim of this study was to assess Y2R as a potential therapeutic target for neuroblastoma. In vitro, Y2R antagonist (BIIE0246) prevented activation of p44/42 mitogen-activated protein kinase (MAPK) induced by endogenous NPY, which resulted in decreased proliferation and induction of Bim-mediated apoptosis. Similar growth-inhibitory effects were achieved with NPY small interfering RNA (siRNA) and Y2R siRNA. In vivo, Y2R antagonist significantly inhibited growth of SK-N-BE(2) and SK-N-AS xenografts, which was associated with decreased activation of p44/42 MAPK, as well as reduced proliferation (Ki67) and increased apoptosis (TdT-mediated dUTP nick end labeling; TUNEL). The Y2R antagonist also exerted an antiangiogenic effect. In vitro, it reduced the proliferation of endothelial cells induced by neuroblastoma-conditioned media. Consequently, the Y2R antagonist-treated xenografts had decreased vascularization and a high degree of focal fibrosis. In human neuroblastoma tissues, the expression of Y2R was observed in both tumor and endothelial cells, while NPY was predominantly expressed in neuroblastoma cells. In summary, Y2R is a promising new target for neuroblastoma therapy affecting both cancer cells and tumor vasculature.

Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor.

The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically.

BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells.

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

Beta-platelet-derived growth factor receptor mediates motility and growth of Ewing's sarcoma cells.

The Ewing's sarcoma family of tumors (ESFT) contain a translocation, t(11;22), which results in the novel oncogenic fusion protein EWS/FLI1. Platelet-derived growth factors (PDGF) and their receptors (PDGFR) are involved in the induction and proliferation of numerous solid tumors and are the potential candidates for novel targeted antitumor therapy. Since a relation was reported between PDGF-C and EWS/FLI1, we sought to characterize the PDGF signaling pathway in ESFT. Eight out of nine ESFT cell lines were found to express significant levels of beta-PDGFR. Interestingly, none of the tested cell lines expressed alpha-PDGFR, which is the receptor isotype required for PDGF-C binding. By immunohistochemical staining 47 of 52 (90.4%) archival tumor samples from patients with ESFT were positive for beta-PDGFR. ESFT cell lines were treated with PDGF-AA or PDGF-BB ligands to evaluate downstream signaling. Autophosphorylation of beta-PDGFR and tyrosine phosphorylation of PLC-gamma, PI3Kp85 and Shc were detected only in PDGF-BB-stimulated cells that express beta-PDGFR. Receptor function was further evaluated using chemotaxis assays that showed TC-32 cell migration towards PDGF-BB. A specific PDGFR kinase inhibitor AG1295 blocked beta-PDGFR activation, downstream signaling, growth in cell culture and chemotaxis of TC-32 cells. AG1295 also delayed tumor formation and prolonged survival in an ESFT animal model. We conclude that ESFT express beta-PDGFR and that this is a functional and potentially crucial signaling pathway. Therefore, beta-PDGFRs may provide a novel therapeutic target in ESFT that can be utilized to design better treatment modalities.

Insulin-like growth factor type 1 (IGF-1) and IGF binding protein-3 in patients with Ewing sarcoma family of tumors.

BACKGROUND: Ewing sarcoma family of tumors (ESFTs) are the second most common bone tumor, that most often affects persons ages 3-40 years. The ESFTs rely on signaling through the insulin-like growth factor-1 receptor (IGF-1R) for growth and transformation. The current studies were performed to determine the levels of IGF-1 and IGF binding protein-3 (IGFBP-3) in patients with ESFT. The authors then performed an exploratory analysis to evaluate whether IGF parameters could differentiate event free or overall survival in ESFT patients. METHODS: The authors measured serum levels of IGF-1 and IGFBP-3 by using a radioimmunoassay from 111 patients with ESFT with a median follow-up of 13 years from diagnosis. RESULTS: The IGF-1 levels were lower among patients with metastatic disease to the bones or the bone marrow compared with patients without metastasis to these sites (p2 = 0.021 and 0.0038, respectively). IGFBP-3 is known to sequester IGF-1; the ratios of IGFBP-3 to IGF-1 were evaluated. Patients with metastatic disease to any site had higher IGFBP-3 to IGF-1 ratios than patients with localized disease (p2 = 0.0067). There was a trend toward increased survival in patients with localized disease who had high IGFBP-3 to IGF-1 levels. Metastatic patients showed a similar trend. CONCLUSIONS: Levels of IGF-1 and IGFBP-3 in ESFT patients can identify patients with the most widespread disease. The IGFBP-3 to IGF-1 ratio in patients with either localized or metastatic disease identified patients with a trend toward increased survival. Further prospective evaluation with higher patient numbers might show a prognostic role for the IGFBP-3 to IGF-1 ratio in patients with ESFT.

Myoepithelioma metastatic to the orbit.

PURPOSE: To report a case of myoepithelioma metastatic to the orbit in an 11-year-old boy. METHODS: Interventional case report. An 11-year-old white male with a history of resection of a left thigh mass 10 months previously presented with a painless, rapid swelling of the left upper eyelid. Computed tomography scan and incisional biopsy of the orbital mass were performed. RESULTS: Immunohistochemical stains of the tumor in the left orbit and the previously resected mass were consistent with myoepithelioma. As a result of widespread metastases, the patient died 4 months after initial presentation to the eye clinic. CONCLUSION: Myoepithelioma should be included in the differential diagnosis of neoplasms that can metastasize to the orbit in the pediatric age group.

Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.

PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens. PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays. RESULTS: MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR. CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.

PBK/TOPK is a novel mitotic kinase which is upregulated in Burkitt's lymphoma and other highly proliferative malignant cells.

PBK/TOPK is a recently cloned serine/threonine kinase which is phosphorylated during mitosis. Earlier work indicated that this kinase is upregulated in a Burkitt's lymphoma cell line (GA-10). To determine whether PBK/TOPK is upregulated in other mitotically active neoplastic cell lines and tissues, Northern analysis was performed on a panel of malignant cell lines and on clinical samples from patients with leukemia or lymphoma. While PBK/TOPK mRNA was not detectable in normal peripheral blood cells and was weakly expressed in hyperplastic tonsillar B-cells, significantly higher levels of mRNA were detected in 8 Burkitt's lymphoma cell lines, 10 other neoplastic cell lines, and 2 clinical samples-one derived from a patient with ALL and a second derived from a patient with relapsed myeloma. In addition, Northern analysis of fetal tissues showed upregulated expression of PBK/TOPK in fetal kidney, lung, spleen, brain, and testis. These data suggest that PBK/TOPK expression is increased in highly proliferative malignant cells and during normal fetal development.

Glypican-3 expression in Wilms tumor and hepatoblastoma.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan. When it is disrupted, it causes the X-linked gigantism-overgrowth Simpson-Golabi-Behmel syndrome. Its involvement in growth control is consistent with recent reports that it can bind to growth factors, possibly including insulin-like growth factor 2. Further, it has been hypothesized that it may function as a tumor suppressor gene in breast and ovarian carcinomas and mesotheliomas. PATIENTS AND METHODS: RNA and protein were extracted from Wilms tumor and hepatoblastoma tissue samples and GPC3 levels were measured in these extracts by Northern blotting, reverse transcription polymerase chain reaction, and immunoblotting. RESULTS: In contrast to published results with carcinomas, high levels of GPC3 expression were found in Wilms tumor and hepatoblastoma. Low or undetectable expressions of this gene were found in normal tissue surrounding the tumor. CONCLUSIONS: Increased expression of GPC3 in Wilms tumor and hepatoblastoma suggests a growth-promoting or neutral activity for this gene product rather than a growth-suppressive effect.

Recognition of common childhood malignancies.

Although cancer has an annual incidence of only about 150 new cases per 1 million U.S. children, it is the second leading cause of childhood deaths. Early detection and prompt therapy have the potential to reduce mortality. Leukemias, lymphomas and central nervous system tumors account for more than one half of new cancer cases in children. Early in the disease, leukemia may cause nonspecific symptoms similar to those of a viral infection. Leukemia should be suspected if persistent vague symptoms are accompanied by evidence of abnormal bleeding, bone pain, lymphadenopathy or hepatosplenomegaly. The presenting symptoms of a brain tumor may include elevated intracranial pressure, nerve abnormalities and seizures. A spinal tumor often presents with signs and symptoms of spinal cord compression. In children, lymphoma may present as one or more painless masses, often in the neck, accompanied by signs and symptoms resulting from local compression, as well as signs and symptoms of systemic disturbances, such as fever and weight loss. A neuroblastoma may arise from sympathetic nervous tissue anywhere in the body, but this tumor most often develops in the abdomen. The presentation depends on the local effects of the solid tumor and any metastases. An abdominal mass in a child may also be due to Wilms' tumor. This neoplasm may present with renal signs and symptoms, such as hypertension, hematuria and abdominal pain. A tumor of the musculoskeletal system is often first detected when trauma appears to cause pain and dysfunction out of proportion to the injury. Primary care physicians should be alert for possible presenting signs and symptoms of childhood malignancy, particularly in patients with Down syndrome or other congenital and familial conditions associated with an increased risk of cancer.

Phosphoinositide 3-hydroxide kinase blockade enhances apoptosis in the Ewing's sarcoma family of tumors.

Ewing's sarcoma family of tumors (ESFTs) affects patients between the ages of 3 and 40 years, with most cases occurring in the second decade of life. These tumors contain a characteristic translocation, t(11;22), that produces a unique fusion protein, EWS/FLI-1. EWS/FLI-1 transforms mouse fibroblasts; this transformation requires intact EWS and FLI-1 domains as well as the insulin-like growth factor-I receptor (IGF-IR). The IGF-IR is a well-described transmembrane tyrosine kinase receptor that modulates transformation, cell growth, and survival. IGF-IR survival signaling is mediated through the downstream activation of phosphoinositide 3-OH kinase (PI 3-K) and Akt. Apoptosis, programmed cell death, progresses from a central death signal to a caspase cascade, including activation of caspase-3. Because the IGF-IR has been shown to play a role in the transformation and growth of ESFTs, we wanted to determine the role of downstream molecules in the cellular response to doxorubicin treatment. Doxorubicin increased caspase-3 activity in two ESFT cell lines, TC-32 and TC-71. Concomitant treatment of the doxorubicin-treated cells with IGF-I reduced caspase-3 activity 8-fold in TC-32 and 4-fold in TC-71. To determine whether PI 3-K has a role in IGF-I-mediated survival in ESFTs, PI 3-K was then inhibited with wortmannin and LY294002. Doxorubicin treatment reduced cell number and enhanced apoptosis in PI 3-K inhibited cells compared with noninhibited cells. Akt, a serine/threonine kinase activated downstream of PI 3-K, was investigated to determine whether its constitutive activation would render ESFT cells more resistant to doxorubicin. A constitutively activated Akt was stably transfected into ESFT and those cells with high levels of expression demonstrated increased resistance to doxorubicin-induced caspase-3 activation. These results indicate that ESFT cell lines use an IGF-IR initiated signaling pathway through PI 3-K and Akt for survival when treated with doxorubicin.

Expression of the wild-type insulin-like growth factor II receptor gene suppresses growth and causes death in colorectal carcinoma cells.

The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.

Cerebral edema and priapism in an adolescent with acute lymphoblastic leukemia.

Priapism and increased intracranial pressure are both rare, but recognized, manifestations of leukemia. However, they have never been reported in the same patient. We report a 15-year-old male with acute lymphoblastic leukemia who presented with hyperleukocytosis, priapism, and increased intracranial pressure. Central nervous system leukostasis and cerebral edema may have been detected earlier, had his history of priapism been known. Management of hyperleukocytosis complicated by priapism and increased intracranial pressure is discussed.

Cytogenetics and experimental models.

The use of cytogenetics has led to significant improvement in the diagnoses and classification of sarcomas. Many of the major sarcomas have been to have characteristic tumor-specific chromosomal translocations that are currently used in the diagnosis of these tumors. In the past year, a subset of Ewing's family of tumors and myxoid liposarcomas, which lack one of the characteristic translocations, were found to carry related translocations. New technologies such as a spectral karyotyping will likely increase out ability to identify additional tumor-specific translocations. The emergence of genetic alterations as prognostic factors, as illustrated by Ewing's family of tumors, osteosarcoma, and p53 expression in soft tissue sarcomas in general, is discussed. The review concludes with laboratory applications derived from either tumor cytogenetic or gene function abnormalities that are related to tumor-specific translocations. It is anticipated that advances in diagnosis, prognosis, and modeling will translate into future therapeutic advances.

Expression of PAX3 in Ewing's sarcoma family of tumors.

The Ewing's sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing's sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material.

Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides.

Ewing's sarcoma family of tumors (EFT) contain reciprocal translocations, of which approximately 90% occur between the long arm of chromosomes 11 and 22,t(11;22)(q24;q12) resulting in the formation of chimeric proteins generated by a fusion of the EWS and FLI-1 genes. To determine if EWS-FLI-1 protein is responsible for the Ewing sarcoma phenotype we have used sequence-specific antisense oligodeoxynucleotides (ODN) to block its expression. We have evaluated a series of antisense ODN directed toward the breakpoint region in an effort to prevent translation of the fusion messenger RNA. ODN were first evaluated in a cell-free in vitro translation system. Exogenously added RNase H was found to be required for translation inhibition. ODN that showed complete inhibition of translation were electroporated into TC-32 cells, a EFT cell line. Fusion protein and EWS protein levels were evaluated by Western blot analysis. A 40-60% decrease in the fusion protein was observed in TC-32 cells with antisense ODN directed toward the breakpoint region. Cell viability was reduced with antisense sequences in TC-32 cells but not in a prostate cancer cell line. Since inhibition of t(11:22) gene product is correlated to effects on cell viability reduction of the fusion protein may thus offer insight into the biology of EFT.

The insulin-like growth factor-I receptor is required for EWS/FLI-1 transformation of fibroblasts.

Ewing's family of tumors is characterized by a well described reciprocal translocation, t(11;22)(q24;q12), which produces a fusion protein (EWS/FLI-1) that transforms mouse fibroblasts. The EWS/FLI-1 fusion protein has been shown to act as a potent chimeric transcription factor. Overexpression of insulin-like growth factor-I receptor (IGF-IR) has been implicated in many tumor models as playing a role in cell growth and tumorigenesis. In addition, blockade of the IGF-IR inhibits the growth of Ewing's family of tumors cells. Therefore, we first studied whether the presence of the IGF-IR is required for transformation by the EWS/FLI-1 fusion protein. To perform this study, we used two previously described fibroblast cell lines, R- and W, derived from an IGF-IR knockout mouse and a wild-type littermate, respectively. Neither W nor R- cells without the fusion protein formed soft agar colonies. However, W clones expressing the fusion message (WF cells) formed soft agar colonies, whereas R- clones expressing the fusion message (R-F cells) did not form soft agar colonies. Because the IGF-IR is required for EWS/FLI-1 transformation, we chose to investigate whether altered signaling occurs from the IGF-IR when the EWS/FLI-1 fusion is present. WF cells demonstrated a greater degree of ligand-stimulated insulin receptor substrate-1 phosphorylation when compared with W cells, suggesting that expression of the EWS/FLI-1 fusion protein alters the IGF-IR signaling pathway.

Involvement of IGF-II in human cancer.

IGF-II is a regulatory peptide which appears to be involved significantly in the progression of many tumors, while minimally involved in post-fetal non-tumor tissue. Interruption of IGF-II pathways therefore offers the possibility of tumor control with a high therapeutic index. Investigators should continue to evaluate tumors for the involvement of IGF-II as well as investigate clinically relevant means of disrupting those pathways.