It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of ...androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.
The pathogenic species of
are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, ...many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of
at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of
species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis.
Opportunistic infections caused by species of the pathogenic yeast
lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of
from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of
or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.
The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of ...PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.
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•Loss of PTEN function cooperates with oncogenic PIK3CA in mammary tumorigenesis•Targeted AKT inhibition suppresses growth of PTEN and PI3K mutant mammary organoids•Loss of PTEN protein-phosphatase activity sensitizes tumors and cells to death•GR mediates the failsafe mechanism driven by loss of PTEN protein function
Yip et al. demonstrate that loss of the tumor suppressor PTEN synergizes with mutant PI3K in mammary tumorigenesis but also renders tumor cells sensitive to death induced by the glucocorticoid receptor (GR). The authors conclude that GR action and AKT inhibition can provide a new treatment for PTEN/PI3K mutant cancers.
Polycystic kidney disease (PKD) is a common cause of renal failure with few effective treatments. INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates phosphoinositide 3-kinase ...(PI3K)-generated PI(3,4,5)P
and is mutated in ciliopathy syndromes. Germline Inpp5e deletion is embryonically lethal, attributed to cilia stability defects, and is associated with polycystic kidneys. However, the molecular mechanisms responsible for PKD development upon Inpp5e loss remain unknown. Here, we show conditional inactivation of Inpp5e in mouse kidney epithelium results in severe PKD and renal failure, associated with a partial reduction in cilia number and hyperactivation of PI3K/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment with an mTORC1 inhibitor improved kidney morphology and function, but did not affect cilia number or length. Therefore, we identify Inpp5e as an essential inhibitor of the PI3K/Akt/mTORC1 signaling axis in renal epithelial cells, and demonstrate a critical role for Inpp5e-dependent mTORC1 regulation in PKD suppression.
Abstract
Polycystic kidney disease (PKD) results from excessive renal epithelial cell proliferation, leading to the formation of large fluid filled cysts which impair renal function and frequently ...lead to renal failure. Hyperactivation of numerous signaling pathways is hypothesized to promote renal epithelial cell hyperproliferation including mTORC1, extracellular signal-regulated kinase (ERK) and WNT signaling. β-catenin and its target genes are overexpressed in some PKD models and expression of activated β-catenin induces cysts in mice; however, β-catenin murine knockout studies indicate it may also inhibit cystogenesis. Therefore, it remains unclear whether β-catenin is pro- or anti-cystogenic and whether its role is canonical WNT signaling-dependent. Here, we investigate whether β-catenin deletion in a PKD model with hyperactived β-catenin signaling affects disease progression to address whether increased β-catenin drives PKD. We used renal epithelial cell specific Inpp5e-null PKD mice which we report exhibit increased β-catenin and target gene expression in the cystic kidneys. Surprisingly, co-deletion of β-catenin with Inpp5e in renal epithelial cells exacerbated polycystic kidney disease and renal failure compared to Inpp5e deletion alone, but did not normalize β-catenin target gene expression. β-catenin/Inpp5e double-knockout kidneys exhibited increased cyst initiation, cell proliferation and MEK/ERK signaling compared to Inpp5e-null, associated with increased fibrosis, which may collectively contribute to accelerated disease. Therefore, increased β-catenin and WNT target gene expression are not necessarily cyst promoting. Rather β-catenin may play a dual and context-dependent role in PKD and in the presence of other cyst-inducing mutations (Inpp5e-deletion); β-catenin loss may exacerbate disease in a WNT target gene-independent manner.
Cancer is a complex and heterogeneous disease marked by the dysregulation of cancer driver genes historically classified as oncogenes or tumour suppressors according to their ability to promote or ...inhibit tumour development and growth, respectively. Certain genes display both oncogenic and tumour suppressor functions depending on the biological context, and as such have been termed dual-role cancer driver genes. However, because of their context-dependent behaviour, the tumourigenic mechanism of many dual-role genes is elusive and remains a significant knowledge gap in our effort to understand and treat cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) is an emerging dual-role cancer driver gene, primarily known for its role as a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway. In response to growth factor stimulation, class I PI3K generates PtdIns(3,4,5)P
at the plasma membrane. PtdIns(3,4,5)P
can be hydrolysed by inositol polyphosphate 5-phosphatases to generate PtdIns(3,4)P
, which, together with PtdIns(3,4,5)P
, facilitates the activation of AKT to promote cell proliferation, survival, migration, and metabolism. Phosphatase and tensin homology on chromosome 10 (PTEN) and INPP4B are dual-specificity phosphatases that hydrolyse PtdIns(3,4,5)P
and PtdIns(3,4)P
, respectively, and thus negatively regulate PI3K/AKT signalling. PTEN is a bona fide tumour suppressor that is frequently lost in human tumours. INPP4B was initially characterised as a tumour suppressor akin to PTEN, and has been implicated as such in a number of cancers, including prostate, thyroid, and basal-like breast cancers. However, evidence has since emerged revealing INPP4B as a paradoxical oncogene in several malignancies, with increased INPP4B expression reported in AML, melanoma and colon cancers among others. Although the tumour suppressive function of INPP4B has been mostly ascribed to its ability to negatively regulate PI3K/AKT signalling, its oncogenic function remains less clear, with proposed mechanisms including promotion of PtdIns(3)P-dependent SGK3 signalling, inhibition of PTEN-dependent AKT activation, and enhancing DNA repair mechanisms to confer chemoresistance. Nevertheless, research is ongoing to identify the factors that dictate the tumourigenic output of INPP4B in different human cancers. In this review we discuss the dualistic role that INPP4B plays in the context of cancer development, progression and treatment, drawing comparisons to PTEN to explore how their similarities and, importantly, their differences may account for their diverging roles in tumourigenesis.
Reducing body myopathy (RBM) is a rare disorder causing progressive muscular weakness characterized by aggresome-like inclusions in the myofibrils. Identification of genes responsible for RBM by ...traditional genetic approaches has been impossible due to the frequently sporadic occurrence in affected patients and small family sizes. As an alternative approach to gene identification, we used laser microdissection of intracytoplasmic inclusions identified in patient muscle biopsies, followed by nanoflow liquid chromatography-tandem mass spectrometry and proteomic analysis. The most prominent component of the inclusions was the Xq26.3-encoded four and a half LIM domain 1 (FHL1) protein, expressed predominantly in skeletal but also in cardiac muscle. Mutational analysis identified 4 FHL1 mutations in 2 sporadic unrelated females and in 2 families with severely affected boys and less-affected mothers. Transfection of kidney COS-7 and skeletal muscle C2C12 cells with mutant FHL1 induced the formation of aggresome-like inclusions that incorporated both mutant and wild-type FHL1 and trapped other proteins in a dominant-negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de novo mutations in FHL1, thereby defining a new X-linked protein aggregation disorder of muscle.
Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, ...including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement.
Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in ...>40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.
The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway regulates many cellular functions including proliferation, migration, survival and protein synthesis. Somatic mutations in PIK3CA, the gene ...encoding the p110α catalytic subunit of PI3K enzyme, are commonly associated with many human cancers as well as recently being implicated in human overgrowth syndromes. However, it is not clear if such mutations can be inherited through the germline. We have used a novel mouse model with Cre recombinase (Cre)-conditional knock-in of the common H1047R mutation into the endogenous Pik3ca gene. Heterozygous expression of the Pik3ca(H1047R) mutation in the developing mouse embryo resulted in failed 'turning' of the embryo and disrupted vascular remodelling within the embryonic and extraembryonic tissues, leading to lethality prior to E10. As vascular endothelial growth factor A (VEGF-A) signalling was disrupted in these embryos, we used Cre under the control of the Tie2 promoter to target the Pik3ca(H1047R) mutation specifically to endothelial cells. In these embryos turning occurred normally but the vascular remodelling defects and embryonic lethality remained, likely as a result of endothelial hyperproliferation. Our results confirm the lethality associated with heterozygous expression of the Pik3ca(H1047R) mutation during development and likely explain the lack of inherited germline PIK3CA mutations in humans.
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