bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2019‒11‒24
fifteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute


  1. Int J Mol Sci. 2019 Nov 18. pii: E5792. [Epub ahead of print]20(22):
    Hillmann P, Fabbro D.
      The phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway has been implicated as a cancer target. Big pharma players and small companies have been developing small molecule inhibitors of PI3K and/or mTOR since the 1990s. Although four inhibitors have been approved, many open questions regarding tolerability, patient selection, sensitivity markers, development of resistances, and toxicological challenges still need to be addressed. Besides clear oncological indications, PI3K and mTOR inhibitors have been suggested for treating a plethora of different diseases. In particular, genetically induced PI3K/mTOR pathway activation causes rare disorders, known as overgrowth syndromes, like PTEN (phosphatase and tensin homolog) hamartomas, tuberous sclerosis complex (TSC), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth spectrum (PROS), and activated PI3-Kinase delta syndrome (PI3KCD, APDS). Some of those disorders likeTSC or hemimegalencephaly, which are one of the PROS disorders, also belong to a group of diseases called mTORopathies. This group of syndromes presents with additional neurological manifestations associated with epilepsy and other neuropsychiatric symptoms induced by neuronal mTOR pathway hyperactivation. While PI3K and mTOR inhibitors have been and still are intensively tested in oncology indications, their use in genetically defined syndromes and mTORopathies appear to be promising avenues for a pharmacological intervention.
    Keywords:  APDS; PI3K; PROS; PTEN hamartoma; TSC; brain penetration; cancer; mTOR inhibitor; mTORopathy; overgrowth syndrome
    DOI:  https://doi.org/10.3390/ijms20225792
  2. Endocrinology. 2019 Nov 21. pii: bqz021. [Epub ahead of print]
    Neff AM, Yu J, Taylor RN, Bagchi IC, Bagchi MK.
      Decidualization, the process by which fibroblastic human endometrial stromal cells (HESC) differentiate into secretory decidual cells, is a critical event during the establishment of pregnancy. It is dependent on the steroid hormone progesterone acting through the nuclear progesterone receptor (PR). Previously, we identified insulin receptor substrate 2 (IRS2) as a factor that is directly regulated by PR during decidualization. IRS2 is an adapter protein that functionally links receptor tyrosine kinases, such as insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R), and their downstream effectors. IRS2 expression was induced in HESC during in vitro decidualization and siRNA-mediated down-regulation of IRS2 transcripts resulted in attenuation of this process. Further use of siRNAs targeted to IR or IGF1R transcripts showed that down-regulation of IR, but not IGF1R, led to impaired decidualization. Loss of IRS2 transcripts in HESC suppressed phosphorylation of both ERK1/2 and AKT, downstream effectors of insulin signaling, which mediate gene expression associated with decidualization and regulate glucose uptake. Indeed, down-regulation of IRS2 resulted in reduced expression and membrane localization of glucose transporters GLUT1 and GLUT4, resulting in lowered glucose uptake during stromal decidualization. Collectively, these data suggest that the PR-regulated expression of IRS2 is necessary for proper insulin signaling controlling gene expression and glucose utilization, which critically support the decidualization process to facilitate pregnancy. This study provides new insight into the mechanisms by which steroid hormone signaling intersects with insulin signaling in the uterus during decidualization, which has important implications for pregnancy complications associated with insulin resistance and infertility.
    Keywords:  Decidualization; Glucose transport; Insulin signaling; Metabolism
    DOI:  https://doi.org/10.1210/endocr/bqz021
  3. Nucleic Acids Res. 2019 Nov 19. pii: gkz1103. [Epub ahead of print]
    Sharma A, Alswillah T, Kapoor I, Debjani P, Willard B, Summers MK, Gong Z, Almasan A.
      Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are predominantly repaired by non-homologous end joining (NHEJ). IR-induced DNA damage activates autophagy, an intracellular degradation process that delivers cytoplasmic components to the lysosome. We identified the deubiquitinase USP14 as a novel autophagy substrate and a regulator of IR-induced DNA damage response (DDR) signaling. Inhibition of autophagy increased levels and DSB recruitment of USP14. USP14 antagonized RNF168-dependent ubiquitin signaling and downstream 53BP1 chromatin recruitment. Here we show that autophagy-deficient cells are defective in NHEJ, as indicated by decreased IR-induced foci (IRIF) formation by pS2056-, pT2609-DNA-PKcs, pS1778-53BP1, RIF1 and a reporter assay activation. Moreover, chromatin recruitment of key NHEJ proteins, including, Ku70, Ku80, DNA-PKcs and XLF was diminished in autophagy-deficient cells. USP14 inhibition rescued the activity of NHEJ-DDR proteins in autophagy-deficient cells. Mass spectrometric analysis identified USP14 interaction with core NHEJ proteins, including Ku70, which was validated by co-immunoprecipitation. An in vitro assay revealed that USP14 targeted Ku70 for deubiquitination. AKT, which mediates Ser432-USP14 phosphorylation, was required for IRIF formation by USP14. Similar to USP14 block, AKT inhibition rescued the activity of NHEJ-DDR proteins in autophagy- and PTEN-deficient cells. These findings reveal a novel negative PTEN/Akt-dependent regulation of NHEJ by USP14.
    DOI:  https://doi.org/10.1093/nar/gkz1103
  4. Circ Res. 2019 Nov 19.
    Chen Z, Yu H, Shi X, Warren CR, Lotta LA, Friesen M, Meissner T, Langenberg C, Wabitsch M, Wareham N, Benson MD, Gerszten RE, Cowan CA.
      Rationale: Genome-wide association studies (GWAS) have identified genetic loci associated with insulin resistance (IR) but pinpointing the causal genes of a risk locus has been challenging. Objective: To identify candidate causal genes for IR, we screened regional and biologically plausible genes (16 in total) near the top ten IR-loci in risk-relevant cell types, namely preadipocytes and adipocytes. Methods and Results: We generated 16 human Simpson-Golabi-Behmel syndrome preadipocyte knockout lines (SGBS-KO) by lentivirus-mediated CRISPR/Cas9 system. We evaluated each gene knockout by screening IR-relevant phenotypes in the three insulin-sensitizing mechanisms, including adipogenesis, lipid metabolism and insulin signaling. We performed genetic analyses to evaluate whether candidate genes prioritized by our in vitro studies were eQTL genes in human subcutaneous adipose tissue, and were associated with risk of IR, type 2 diabetes (T2D) and cardiovascular diseases (CVD). We further validated the functions of three new adipose IR genes by phenotypic rescue in the SGBS-KO cell lines. Results: Twelve genes, PPARG, IRS-1, FST, PEPD, PDGFC, MAP3K1, GRB14, ARL15, ANKRD55, RSPO3, COBLL1 and LYPLAL1, showed diverse phenotypes in the three insulin-sensitizing mechanisms, and the first seven of these genes could affect all the three mechanisms. Five of six eQTL genes are among the top candidate causal genes and the abnormal expression levels of these genes (IRS-1, GRB14, FST, PEPD and PDGFC) in human SAT could be associated with increased risk of IR, T2D and CVD. Phenotypic rescue of FST, PEPD and PDGFC in the SGBS-KO lines confirmed their function in adipose IR. Conclusions: Twelve genes showed diverse phenotypes indicating differential roles in insulin sensitization, suggesting mechanisms bridging the association of their genomic loci with IR. We prioritized PPARG, IRS-1, GRB14, MAP3K1, FST, PEPD and PDGFC as top candidate genes. Our work points to novel roles for FST, PEPD and PDGFC in adipose tissue, with consequences for cardiometabolic diseases.
    Keywords:  CRISPR/Cas9 system; Insulin resistance; adipogenesis; insulin signaling; lipid metabolism
    DOI:  https://doi.org/10.1161/CIRCRESAHA.119.315246
  5. Sci Rep. 2019 Nov 18. 9(1): 16992
    Budi EH, Hoffman S, Gao S, Zhang YE, Derynck R.
      Insulin signaling governs many processes including glucose homeostasis and metabolism, and is therapeutically used to treat hyperglycemia in diabetes. We demonstrated that insulin-induced Akt activation enhances the sensitivity to TGF-β by directing an increase in cell surface TGF-β receptors from a pool of intracellular TGF-β receptors. Consequently, increased autocrine TGF-β signaling in response to insulin participates in insulin-induced angiogenic responses of endothelial cells. With TGF-β signaling controlling many cell responses, including differentiation and extracellular matrix deposition, and pathologically promoting fibrosis and cancer cell dissemination, we addressed to which extent autocrine TGF-β signaling participates in insulin-induced gene responses of human endothelial cells. Transcriptome analyses of the insulin response, in the absence or presence of a TGF-β receptor kinase inhibitor, revealed substantial positive and negative contributions of autocrine TGF-β signaling in insulin-responsive gene responses. Furthermore, insulin-induced responses of many genes depended on or resulted from autocrine TGF-β signaling. Our analyses also highlight extensive contributions of autocrine TGF-β signaling to basal gene expression in the absence of insulin, and identified many novel TGF-β-responsive genes. This data resource may aid in the appreciation of the roles of autocrine TGF-β signaling in normal physiological responses to insulin, and implications of therapeutic insulin usage.
    DOI:  https://doi.org/10.1038/s41598-019-53490-x
  6. Curr Diab Rep. 2019 Nov 20. 19(11): 138
    Hsiao WY, Guertin DA.
      PURPOSE OF REVIEW: Obesity is a major risk factor for type 2 diabetes. Although adipose tissue allows storage of excess calories in periods of overnutrition, in obesity, adipose tissue metabolism becomes dysregulated and can promote metabolic diseases. This review discusses recent advances in understandings how adipocyte metabolism impacts metabolic homeostasis.RECENT FINDINGS: The ability of adipocytes to synthesize lipids from glucose is a marker of metabolic fitness, e.g., low de novo lipogenesis (DNL) in adipocytes correlates with insulin resistance in obesity. Adipocyte DNL may promote synthesis of special "insulin sensitizing" signaling lipids that act hormonally. However, each metabolic intermediate in the DNL pathway (i.e., citrate, acetyl-CoA, malonyl-CoA, and palmitate) also has second messenger functions. Mounting evidence suggests these signaling functions may also be important for maintaining healthy adipocytes. While adipocyte DNL contributes to lipid storage, lipid precursors may have additional second messenger functions critical for maintaining adipocyte health, and thus systemic metabolic homeostasis.
    Keywords:  Acetyl-CoA; Citrate; Insulin resistance; Malonyl-CoA; Obesity; Palmitate
    DOI:  https://doi.org/10.1007/s11892-019-1264-9
  7. J Biol Chem. 2019 Nov 19. pii: jbc.RA119.011695. [Epub ahead of print]
    Krycer JR, Elkington SD, Diaz-Vegas A, Cooke KC, Burchfield JG, Fisher-Wellman KH, Cooney GJ, Fazakerley DJ, James DE.
      Insulin action in adipose tissue is crucial for whole-body glucose homeostasis, with insulin resistance being a major risk factor for metabolic diseases such as type 2 diabetes. Recent studies have proposed mitochondrial oxidants as a unifying driver of adipose insulin resistance, serving as a signal of nutrient excess. However, neither the substrates for nor sites of oxidant production are known. Since insulin stimulates glucose utilisation, we hypothesised that glucose oxidation would fuel respiration, in turn generating mitochondrial oxidants. This would impair insulin action, limiting further glucose uptake in a negative feedback loop of 'glucose-dependent' insulin resistance. Using primary rat adipocytes and cultured 3T3-L1 adipocytes, we observed that insulin increased respiration, but notably this occurred independently of glucose supply. In contrast, glucose was required for insulin to increase mitochondrial oxidants. Despite rising to similar levels as when treated with other agents that cause insulin resistance, glucose-dependent mitochondrial oxidants failed to cause insulin resistance. Subsequent studies revealed a temporal relationship whereby mitochondrial oxidants needed to increase before the insulin stimulus to induce insulin resistance. Together, these data reveal that a) adipocyte respiration is principally fuelled from non-glucose sources, b) there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present, and c) mitochondrial oxidative stress must precede the insulin stimulus to cause insulin resistance, explaining why short-term insulin-dependent glucose utilisation does not promote insulin resistance. These data provide additional clues to mechanistically link nutrient excess to adipose insulin resistance.
    Keywords:  adipocyte; glucose; insulin; insulin resistance; mitochondria; oxidative stress; respiration
    DOI:  https://doi.org/10.1074/jbc.RA119.011695
  8. Cell Commun Signal. 2019 Nov 21. 17(1): 154
    Hinz N, Jücker M.
      BACKGROUND: AKT, also known as protein kinase B, is a key element of the PI3K/AKT signaling pathway. Moreover, AKT regulates the hallmarks of cancer, e.g. tumor growth, survival and invasiveness of tumor cells. After AKT was discovered in the early 1990s, further studies revealed that there are three different AKT isoforms, namely AKT1, AKT2 and AKT3. Despite their high similarity of 80%, the distinct AKT isoforms exert non-redundant, partly even opposing effects under physiological and pathological conditions. Breast cancer as the most common cancer entity in women, frequently shows alterations of the PI3K/AKT signaling.MAIN CONTENT: A plethora of studies addressed the impact of AKT isoforms on tumor growth, metastasis and angiogenesis of breast cancer as well as on therapy response and overall survival in patients. Therefore, this review aimed to give a comprehensive overview about the isoform-specific effects of AKT in breast cancer and to summarize known downstream and upstream mechanisms. Taking account of conflicting findings among the studies, the majority of the studies reported a tumor initiating role of AKT1, whereas AKT2 is mainly responsible for tumor progression and metastasis. In detail, AKT1 increases cell proliferation through cell cycle proteins like p21, p27 and cyclin D1 and impairs apoptosis e.g. via p53. On the downside AKT1 decreases migration of breast cancer cells, for instance by regulating TSC2, palladin and EMT-proteins. However, AKT2 promotes migration and invasion most notably through regulation of β-integrins, EMT-proteins and F-actin. Whilst AKT3 is associated with a negative ER-status, findings about the role of AKT3 in regulation of the key properties of breast cancer are sparse. Accordingly, AKT1 is mutated and AKT2 is amplified in some cases of breast cancer and AKT isoforms are associated with overall survival and therapy response in an isoform-specific manner.
    CONCLUSIONS: Although there are several discussed hypotheses how isoform specificity is achieved, the mechanisms behind the isoform-specific effects remain mostly unrevealed. As a consequence, further effort is necessary to achieve deeper insights into an isoform-specific AKT signaling in breast cancer and the mechanism behind it.
    Keywords:  AKT; Breast cancer; Isoforms; PI3K/AKT signaling; Protein kinase B
    DOI:  https://doi.org/10.1186/s12964-019-0450-3
  9. Mol Cell. 2019 Nov 06. pii: S1097-2765(19)30794-4. [Epub ahead of print]
    Odle RI, Walker SA, Oxley D, Kidger AM, Balmanno K, Gilley R, Okkenhaug H, Florey O, Ktistakis NT, Cook SJ.
      Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.
    Keywords:  ATG13; ATG14; CDK1; RAPTOR; TFEB; ULK1; autophagy; mTOR; mitosis
    DOI:  https://doi.org/10.1016/j.molcel.2019.10.016
  10. Biochim Biophys Acta Gene Regul Mech. 2019 Nov 18. pii: S1874-9399(18)30452-8. [Epub ahead of print] 194449
    Aboudehen K.
      The mechanistic target of rapamycin (mTOR) is a major signaling hub that coordinates cellular and organismal responses, such as cell growth, proliferation, apoptosis, and metabolism. Dysregulation of mTOR signaling occurs in many human diseases, and there are significant ongoing efforts to pharmacologically target this pathway. Long noncoding RNAs (lncRNA), defined by a length > 200 nucleotides and absence of a long open-reading-frame, are a class of non-protein-coding RNAs. Mutations and dysregulations of lncRNAs are directly linked to the development and progression of many diseases, including cancer, diabetes, and neurologic disorders. Recent findings reveal diverse functions for lncRNA that include transcriptional regulation, organization of nuclear domains, and regulation of proteins or RNA molecules. Despite considerable development in our understanding of lncRNA over the past decade, only a fraction of annotated lncRNAs has been examined for biological function. In addition, lncRNAs have emerged as therapeutic targets due to their ability to modulate multiple pathways, including mTOR signaling. This review will provide an up-to-date summary of lncRNAs that are involved in regulating mTOR pathway.
    DOI:  https://doi.org/10.1016/j.bbagrm.2019.194449
  11. Commun Biol. 2019 ;2 414
    Sinkala M, Mulder N, Patrick Martin D.
      Malignant cells reconfigure their metabolism to support oncogenic processes such as accelerated growth and proliferation. The mechanisms by which this occurs likely involve alterations to genes that encode metabolic enzymes. Here, using genomics data for 10,528 tumours of 32 different cancer types, we characterise the alterations of genes involved in various metabolic pathways. We find that mutations and copy number variations of metabolic genes are pervasive across all human cancers. Based on the frequencies of metabolic gene alterations, we further find that there are two distinct cancer supertypes that tend to be associated with different clinical outcomes. By utilising the known dose-response profiles of 825 cancer cell lines, we infer that cancers belonging to these supertypes are likely to respond differently to various anticancer drugs. Collectively our analyses define the foundational metabolic features of different cancer supertypes and subtypes upon which discriminatory strategies for treating particular tumours could be constructed.
    Keywords:  Biochemical reaction networks; Cancer genomics; Data integration; Tumour heterogeneity
    DOI:  https://doi.org/10.1038/s42003-019-0666-1
  12. Biochem J. 2019 Nov 18. pii: BCJ20190665. [Epub ahead of print]
    Krndija D, Fairhead M.
      Filopodia are thin, actin-based membrane protrusions with roles in sensing external mechanical and chemical cues, such as growth factor gradients in tissues. It was proposed that the chemical sensing role of filopodia is achieved through clearance of activated signaling receptors from filopodia. Type I insulin-like growth factor receptor (IGF1R) is a key regulator of normal development and growth, as well as tumor development and progression. Its biological roles depend on its activation upon IGF1 binding at the cell membrane. IGF1R behavior at the cell membrane and in particular in filopodia, has not been established. We found that IGF1 activation led to a gradual reduction in IGF1R puncta in filopodia, and that this clearance depended on actin, non-muscle myosin II, and IGF1R kinase activity. Using single particle tracking of filopodial IGF1R, we established that ligand-free IGF1R undergoes non-directional unidimensional diffusion along the filopodium. Moreover, after initial diffusion, the ligand-bound IGF1R is actively transported along the filopodium towards the filopodium base, and consequently cleared from the filopodium. Our results show that IGF1R can move directionally on the plasma membrane protrusions, supporting a sensory role for filopodia in interpreting local IGF1 gradients.
    Keywords:  filopodia; insulin-like growth factor; receptors; trafficking
    DOI:  https://doi.org/10.1042/BCJ20190665
  13. Elife. 2019 Nov 19. pii: e50036. [Epub ahead of print]8
    Palmer AC, Chidley C, Sorger PK.
      Curative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R‑CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R‑CHOP exhibit very low cross-resistance but not synergistic interaction: together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50-year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.
    Keywords:  cancer biology; computational biology; human; systems biology
    DOI:  https://doi.org/10.7554/eLife.50036
  14. Curr Drug Targets. 2019 Nov 20.
    Lengyel CG, Altuna SC, Habeeb BS, Trapani D, Khan SZ.
      AIMS: In this narrative review, we summarize the role and significance of PI3K-AKT-mTOR (PAM) pathway in ovarian and endometrial cancers, providing the most recent and relevant literature on the topic and addressing options for targeting PAM along with future perspectives of drug development.BACKGROUND: Alterations of the PAM-pathway are common in both endometrial and ovarian cancers, and are described in specific histology-defined subtypes. PAM seems to be involved in critical steps of endometrial and ovarian carcinogenesis, often mechanistically involved in the acquisition of a phenotype of treatment resistance, which could be targetable. However, early clinical trials with PAM-inhibitors (PAMi) have provided disappointing results, particularly when non isoform-specific inhibitors were tested in unselected populations, accompanied by an adverse safety profile. Since then, more encouraging observations have been collected when targeting specific isoforms of PAM proteins with more selective drugs, resulting in encouraging activity and more manageable toxicity.
    CONCLUSION: Although the rational of inhibiting the PAM-pathway has been demonstrated in several promising preclinical studies, no Phase III clinical trial is available to demonstrate a significant benefit of PAM-inhibitors. A way to manage targeted agents is to tailor their use to particular subpopulations most likely to obtain a considerable benefit, namely pursuing an individualized, precision-medicine approach.
    Keywords:  Endometrial Cancer; Gynaecological Cancer; Ovarian Cancer; PI3K-AKT-Mtor Pathway; Platinum Resistance; Precision Medicine; Targeted Therapy
    DOI:  https://doi.org/10.2174/1389450120666191120123612
  15. Oncotarget. 2019 Nov 05. 10(60): 6526-6535
    Piha-Paul SA, Taylor MH, Spitz D, Schwartzberg L, Beck JT, Bauer TM, Meric-Bernstam F, Purkayastha D, Karpiak L, Szpakowski S, Braiteh F.
      Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation plays a key role in tumorigenesis and has been associated with poor prognosis and resistance to multiple therapies in various cancers. Results: There were 146 patients enrolled; common tumor types were colorectal, sarcoma, and ovarian. Tumors had PI3K pathway alterations and a median of four mutations with tissue-specific patterns of mutation burden (lowest: sarcoma [2.5]; highest: esophagus, germ cell tumor, skin non-melanoma, vaginal [7]). The number of prior therapies did not correlate with the number of genetic alterations (Pearson r = -0.037). The clinical benefit rate was 15.1% (n = 22). An additional patient had an unconfirmed complete response. The most common adverse events were fatigue, nausea, hyperglycemia, decreased appetite, and diarrhea. Patient and Methods: In this phase 2, open-label, single-arm study, patients with solid or hematologic malignancies with PI3K pathway activation and progression on or after standard treatment received buparlisib (100 mg once daily). The primary endpoint was clinical benefit rate per local investigator assessment (response or stable disease at ≥16 weeks). Conclusions: Buparlisib was well tolerated, however efficacy was limited despite selection of PI3K pathway aberrations. Future studies may provide insight into buparlisib efficacy by refining the molecular selection of different tumor types.
    Keywords:  advanced malignancies; buparlisib; molecular selection; phosphatidylinositol 3-kinase pathway; tissue agnostic
    DOI:  https://doi.org/10.18632/oncotarget.27251