A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples.

Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed, paraffin-embedded material. Here, we present a methodology [pathology tissue-ChIP (PAT-ChIP)] to extract and immunoprecipitate chromatin from paraffin-embedded patient samples up to several years old. In a pairwise comparison with canonical ChIP, PAT-ChIP showed a high reproducibility of results for several histone marks and an identical ability to detect dynamic changes in chromatin structure upon pharmacological treatment. Finally, we showed that PAT-ChIP can be coupled with high-throughput sequencing (PAT-ChIP-Seq) for the genome-wide analysis of distinct chromatin modifications. PAT-ChIP therefore represents a versatile procedure and diagnostic tool for the analysis of epigenetic alterations in cancer and potentially other diseases.

Cell stretching activates an ATM mechano-transduction pathway that remodels cytoskeleton and chromatin.

Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.

Inactivation of the p53 pathway in retinoblastoma.

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

LSD1-directed therapy affects glioblastoma tumorigenicity by deregulating the protective ATF4-dependent integrated stress response.

Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, poor blood-brain barrier penetration, and resistance to therapy highlight the need for new targets and clinical treatments. A step toward clinical translation includes the eradication of GBM tumor-initiating cells (TICs), responsible for GBM heterogeneity and relapse. By using patient-derived TICs and xenograft orthotopic models, we demonstrated that the selective lysine-specific histone demethylase 1 inhibitor DDP_38003 (LSD1i) is able to penetrate the brain parenchyma in vivo in preclinical models, is well tolerated, and exerts antitumor activity in molecularly different GBMs. LSD1 genetic targeting further strengthens the role of LSD1 in GBM TIC maintenance. GBM TIC plasticity supports their adaptation and survival under a plethora of environmental stresses, including nutrient deficiency and proteostasis perturbation. By mimicking these stresses in vitro, we found that LSD1 inhibition hampers the induction of the activating transcription factor 4 (ATF4), the master regulator of the integrated stress response (ISR). The resulting aberrant ISR sensitizes GBM TICs to stress-induced cell death, hampering tumor aggressiveness. Functionally, LSD1i interferes with LSD1 scaffolding function and prevents its interaction with CREBBP, a critical ATF4 activator. By disrupting the interaction between CREBBP and LSD1-ATF4 axis, LSD1 inhibition prevents GBM TICs from overcoming stress and sustaining GBM progression. The effectiveness of the LSD1 inhibition in preclinical models shown here places a strong rationale toward its clinical translation for GBM treatment.

Expression of the transcription factor HEY1 in glioblastoma: a preliminary clinical study.

AIMS AND BACKGROUND: The hairy/enhancer of split (E(spl))-related family of transcription factors (HES and HEY) are established targets of the notch signaling pathway, which has been implicated in different developmental processes, tumor formation and the self-renewal of neural stem cells. We determined the expression of HEY1 in human malignant gliomas to investigate whether its expression might be related to prognosis. METHODS: The expression of HEY1 was studied by in situ hybridization on 62 cases of glioblastoma. Patients were treated with surgery followed by chemotherapy and radiotherapy. We considered as end points of the study the overall survival time and progression-free interval. Correlations between HEY1 expression and tumor grade/patient overall survival and free interval before recurrence were analyzed using univariate analysis. RESULTS: Based on the in situ hybridization results, HEY1 expression rate was reported as negative staining in 13 cases (20.6%), as weak staining in 11 cases (17.3%), as moderate staining in 21 cases (33.3%), and as strong staining in 17 cases. We considered in the analysis the cumulative expression of HEY1 at in situ hybridization (Hey Index) as negative in 13 cases and positive in 49 cases (77.78%). The overall survival (P = 0.002) and the free-interval (P = 0.012) were significantly longer in patients who were negative for HEY1 expression. CONCLUSIONS: Our data suggest that expression of HEY1 might be used as a marker to distinguish glioblastoma patients with a relatively good prognosis from those at high-risk, and that, in the future, HEY1 might represent a therapeutic target.

A role for the transcription factor HEY1 in glioblastoma.

Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

RaLP, a new member of the Src homology and collagen family, regulates cell migration and tumor growth of metastatic melanomas.

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.

Long-lasting induction of Notch2 in the hippocampus of kainate-treated adult mice.

Notch proteins are involved in cell fate specification during development in tissues including brain. Little is known about their function in adulthood. Recently, Notch receptors have been hypothesized to play a role in neurodegeneration and in particular in Alzheimer's disease (Notch1) and CADASIL (Notch3). Here we show that another family member (Notch2) is constitutively expressed in adult mouse hippocampus in DG and not in CA1 and CA3 neurons. Treatment with kainic acid resulted in marked Notch2 induction in pyramidal neurons of CA1 and in a subpopulation of CA3 neurons surviving the lesion and protein expression was still detectable 6 weeks after drug treatment. These results suggest Notch2 involvement in the response of postmitotic neurons to excitotoxic stimuli.

Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop.

New cancer therapies are likely to arise from an in-depth understanding of the signaling networks influencing tumor initiation, progression and metastasis. We show a fundamental role for Src-homology 2 domain-containing phosphatase 2 (SHP2) in these processes in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancers. Knockdown of SHP2 eradicated breast tumor-initiating cells in xenograft models, and SHP2 depletion also prevented invasion in three-dimensional cultures and in a transductal invasion assay in vivo. Notably, SHP2 knockdown in established breast tumors blocked their growth and reduced metastasis. Mechanistically, SHP2 activated stemness-associated transcription factors, including v-myc myelocytomatosis viral oncogene homolog (c-Myc) and zinc finger E-box binding homeobox 1 (ZEB1), which resulted in the repression of let-7 microRNA and the expression of a set of 'SHP2 signature' genes. We found these genes to be simultaneously activated in a large subset of human primary breast tumors that are associated with invasive behavior and poor prognosis. These results provide new insights into the signaling cascades influencing tumor-initiating cells as well as a rationale for targeting SHP2 in breast cancer.

MMSET is highly expressed and associated with aggressiveness in neuroblastoma.

MMSET (WHSC1/NSD2) is a SET domain-containing histone lysine methyltransferase the expression of which is deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation associated with poor prognosis. Recent studies have shown that MMSET mRNA levels are increased in other tumor types as well. We have carried out immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n = 164). The expression level of MMSET in neuroblastomas was significantly associated with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and postchemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated after treatment, we show that retinoic acid-induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we show that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation. Importantly, we show that MMSET is required for proliferation of neuroblastoma cells and brain-derived neural stem cells. Taken together, our results suggest that MMSET is implicated in neuroblastomagenesis possibly by supporting proliferation of progenitor cells and negatively regulating their differentiation. In this respect, MMSET might be a strong candidate therapeutic target in a subset of neuroblastomas with unfavorable prognosis.

ATAD2 is a novel cofactor for MYC, overexpressed and amplified in aggressive tumors.

The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription.

Lap2alpha expression is controlled by E2F and deregulated in various human tumors.

Deregulation of the retinoblastoma (pRB) tumor suppressor pathway is frequently observed in human cancer and associated with aberrant activity of E2F transcription factors. We have performed microarray based analysis with the aim of identifying potential downstream mediators of the tumor suppressing activity of pRB. Here we report that the expression of LAP2 (lamina-associated polypeptide 2) is under direct control of E2F transcription factors. Chromatin immunoprecipitation assays show that the LAP2 promoter is bound by endogenous E2F in vivo. The LAP2 promoter is transactivated by ectopically expressed E2F and mutation of E2F binding sites eliminates this effect. We studied the expression level of LAP2alpha in human tumors by tissue microarray analysis and found LAP2alpha over expression in a significant percentage of primary larynx, lung, stomach, breast, and colon cancer tissues. In agreement with its regulation by E2F, LAP2alpha over expression in primary tumors was found to be correlated with tumor proliferation rate.

DEK Expression is controlled by E2F and deregulated in diverse tumor types.

Deregulation of the retinoblastoma (pRB) tumor suppressor pathway associated with aberrant activity of E2F transcription factors is frequently observed in human cancer. Microarray based analyses have revealed a large number of potential downstream mediators of the tumor suppressing activity of pRB, including DEK, a fusion partner of CAN found in a subset of acute myeloid leukaemia (AML) patients carrying a (6; 9) translocation. Here we report that the expression of DEK is under direct control of E2F transcription factors. Chromatin immunoprecipitation assays show that the DEK promoter is bound by endogenous E2F in vivo. The DEK promoter is transactivated by E2F and mutation of E2F binding sites eliminates this effect. Expression levels of DEK in human tumors have been investigated by tissue micro array analysis. We find that DEK is overexpressed in many solid tumors such as colon cancer, larynx cancer, bladder cancer, and melanoma.

Frequent alterations in the expression of serine/threonine kinases in human cancers.

Protein kinases constitute a large family of regulatory enzymes involved in the homeostasis of virtually every cellular process. Subversion of protein kinases has been frequently implicated in malignant transformation. Within the family, serine/threonine kinases (STK) have received comparatively lesser attention, vis-a-vis tyrosine kinases, in terms of their involvement in human cancers. Here, we report a large-scale screening of 125 STK, selected to represent all major subgroups within the subfamily, on nine different types of tumors ( approximately 200 patients), by using in situ hybridization on tissue microarrays. Twenty-one STK displayed altered levels of transcripts in tumors, frequently with a clear tumor type-specific dimension. We identified three patterns of alterations in tumors: (a) overexpression in the absence of expression in the normal tissues (10 kinases), (b) overexpression in the presence of expression by normal tissues (8 kinases), and (c) underexpression (3 kinases). Selected members of the three classes were subjected to in-depth analysis on larger case collections and showed significant correlations between their altered expression and biological and/or clinical variables. Our findings suggest that alteration in the expression of STK is a relatively frequent occurrence in human tumors. Among the overexpressed kinases, 10 were undetectable in normal controls and are therefore ideal candidates for further validation as potential targets of molecular cancer therapy.

The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.