Monica Venere

Valosin-Containing Protein Phosphorylation at Ser784 in Response to DNA Damage

The response of eukaryotic cells to DNA damage includes the activation of phosphatidylinositol-3 kinase-related kinases (PIKK), such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK). These three kinases have very similar substrate specificities in vitro, but in vivo, their substrates overlap only partially. Several in vivo substrates of ATM and ATR have been identified and almost all of them are involved in DNA damage-induced cell cycle arrest and/or apoptosis. In contrast, few in vivo substrates of DNA-PK have been identified. These include histone H2AX and DNA-PK itself. We identify here valosin-containing protein (VCP) as a novel substrate of DNA-PK and other PIKK family members. VCP is phosphorylated at Ser784 within its COOH terminus, a region previously shown to target VCP to specific intracellular compartments. Furthermore, VCP phosphorylated at Ser784 accumulated at sites of DNA double-strand breaks (DSBs). VCP is a protein chaperone that unfolds and translocates proteins. Its phosphorylation in response to DNA damage and its recruitment to sites of DNA DSBs could indicate a role of VCP in DNA repair.

Phosphorylation of ATR-Interacting Protein on Ser239 Mediates an Interaction with Breast-Ovarian Cancer Susceptibility 1 and Checkpoint Function

The signaling of DNA damage and replication stress involves a multitude of proteins, including the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), and proteins with BRCA1 COOH-terminal (BRCT) domains. The BRCT domain-containing proteins facilitate the phosphorylation of ATM/ATR substrates and can be coimmunoprecipitated with ATM or ATR. However, their mode of interaction with the ATM/ATR kinases remains elusive. Here, we show that breast-ovarian cancer susceptibility 1 (BRCA1) interacts directly with ATR-interacting protein (ATRIP), an obligate partner of ATR. The interaction involves the BRCT domains of BRCA1 and Ser(239) of ATRIP, a residue that is phosphorylated in both irradiated and nonirradiated cells. Consistent with a role of BRCA1 in ATR signaling, substitution of Ser(239) of ATRIP with Ala leads to a G(2)-M checkpoint defect. We propose that a direct physical interaction between BRCA1 and ATRIP is required for the checkpoint function of ATR.

Pharmacological targeting of the histone chaperone complex FACT preferentially eliminates glioblastoma stem cells and prolongs survival in preclinical models

The nearly universal recurrence of glioblastoma (GBM) is driven in part by a treatment-resistant subpopulation of GBM stem cells (GSC). To identify improved therapeutic possibilities, we combined the EGFR/HER2 inhibitor lapatinib with a novel small molecule, CBL0137, which inhibits FACT (facilitates chromatin transcription), a histone chaperone complex predominantly expressed in undifferentiated cells. Lapatinib and CBL0137 synergistically inhibited the proliferation of patient-derived GBM cells. Compared with non-stem tumor cells (NSTC) enriched from the same specimens, the GSCs were extremely sensitive to CBL0137 monotherapy or FACT knockdown. FACT expression was elevated in GSCs compared with matched NSTCs and decreased in GSCs upon differentiation. Acute exposure of GSCs to CBL0137 increased asymmetric cell division, decreased GSC marker expression, and decreased the capacity of GSCs to form tumor spheres in vitro and to initiate tumors in vivo Oral administration of CBL0137 to mice bearing orthotopic GBM prolonged their survival. Knockdown of FACT reduced the expression of genes encoding several core stem cell transcription factors (SOX2, OCT4, NANOG, and OLIG2), and FACT occupied the promoters of these genes. FACT expression was elevated in GBM tumors compared with non-neoplastic brain tissues, portended a worse prognosis, and positively correlated with GSC markers and stem cell gene expression signatures. Preferential targeting of GSCs by CBL0137 and synergy with EGFR inhibitors support the development of clinical trials combining these two agents in GBM. Cancer Res; 76(8); 2432-42. ©2016 AACR.

Coordinated regulation of p31(Comet) and Mad2 expression is required for cellular proliferation.

p31Comet is a well-known interacting partner of the spindle assembly checkpoint (SAC) effector molecule Mad2. At the molecular level it is well established that p31Comet promotes efficient mitotic exit, specifically the metaphase–anaphase transition, by antagonizing Mad2 function. However, there is little knowledge of how p31Comet is regulated or the physiological importance of controlling p31Comet. Here, we show that the Rb–E2F pathway regulates p31Comet expression. In multiple tumor types (including breast and lung) p31Comet expression is increased along with Mad2. Expression of this antagonist–target pair is coordinated in cells and correlated in cancer. Moreover, a narrow range of p31Comet:Mad2 ratios is compatible with cellular viability. Our data suggest that coordinate regulation is important for the outgrowth of oncogenic cell populations. Our findings suggest that altered p31Comet:Mad2 expression ratios may provide new insight into altered SAC function and the generation of chromosomal instability in tumors.

A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function

Significance Members of the kinesin superfamily serve a wide variety of functions, and a dominant narrative for these molecular motors has been that each member of the superfamily is uniquely specialized to serve a very limited set of functions. However, it is now appreciated that many members of this group serve several distinct physiological roles, and it has been unclear how these kinesins accomplish this functional flexibility. In this report, we describe a posttranslational modification of the kinesin 5 family member Eg5 that dramatically alters its chemomechanical behavior to make it function much more efficiently under load and in ensembles. This work provides the biophysical context required to mechanistically understand the effects of modified Eg5 in dividing cells. Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these—acetylation of lysine 146 in Eg5—by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site–neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant—features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a “brake” that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.

PARP inhibition sensitizes colorectal cancer-initiating cells to chemotherapy: PARP Inhibition and Chemosensitivity

Colorectal cancer (CRC) remains a leading killer in the U.S. with resistance to treatment as the largest hurdle to cure. Colorectal cancer‐initiating cells (CICs) are a self‐renewing tumor population that contribute to tumor relapse. Here, we report that patient‐derived CICs display relative chemoresistance compared with differentiated progeny. In contrast, conventional cell lines failed model therapeutic resistance. CICs preferentially repaired chemotherapy‐induced DNA breaks, prompting us to interrogate DNA damage pathways against which pharmacologic inhibitors have been developed. We found that CICs critically depended on the key single‐strand break repair mediator, poly(ADP‐ribose) polymerase (PARP), to survive treatment with standard‐of‐care chemotherapy. Small molecule PARP inhibitors (PARPi) sensitized CICs to chemotherapy and reduced chemotherapy‐treated CIC viability, self‐renewal, and DNA damage repair. Although PARPi monotherapy failed to kill CICs, combined PARPi therapy with chemotherapy attenuated tumor growth in vivo. Clinical significance of PARPi for CRC patients was supported by elevated PARP levels in colorectal tumors compared with normal colon, with further increases in metastases. Collectively, our results suggest that PARP inhibition serves as a point of fragility for CICs by augmenting therapeutic efficacy of chemotherapy. Stem Cells 2019;37:42–53

Apoptosis Pathways and Chemotherapy in Brain Tumors

Abstract Apoptosis, the major programed cell death pathway, is a mechanism of both normal homeostasis and disease. In the context of tumors, the apoptotic machinery is altered to favor tumor expansion despite harsh microenvironmental conditions and therapeutic interventions aimed at inducing cell death. Insights into both the basic and the neoplastic regulation of apoptosis have opened up therapeutic strategies that have already reached the clinic with their full implication yet to be realized. The main objective of this chapter is to review the molecular mechanisms of apoptosis, as well as provide an overview of the existing knowledge of how apoptosis is deregulated in glioblastoma, how it impacts the tumor environment and response to chemotherapy, as well as novel approaches to trigger apoptosis in brain tumors.

USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination

ABSTRACT Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair. Abbreviations: ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.

Not just another biomarker: the role of integrin alpha 7 in glioblastoma

Glioblastoma is the most lethal primary brain tumor, and one of the most aggressive and invasive types of cancer overall. Despite treatment efforts, median length of survival for glioblastoma patients is between 12 and 18 months (1). Aside from their aggressive nature, glioblastomas are also known for being heterogeneous, with many different cell types throughout the tumor. Glioblastoma stem cells (GSCs) are one cellular subtype within these tumors and are characterized by unlimited self-renewal and resistance to treatment. Cancer stem cells have been identified in brain tumors as well as other cancers, and are thought to play a central role in the malignancy of these tumors (2,3). Hence, there is huge interest amongst researchers to find ways to target the cancer stem cell population specifically. Knowing which cells within a tumor are the extremely tumorigenic GSCs is not possible without reliable biomarkers. As mentioned previously, glioblastomas are heterogeneous within each individual tumor, but are also heterogeneous from one patient to the next. Many groups have identified markers of cancer stem cells that turn out to have greater representation in certain subtypes over others, such as CD44 in mesenchymal tumor regions, and CD133/Olig2 in proneural tumor regions (4-7). Despite the wide use of these surface epitopes as biomarkers, there is still controversy over whether or not they are uniformly reliable across patient samples (2).

Foe becomes friend as Zika joins the fight against cancer

Zika virus preferentially targets cancer stem cells in glioblastoma, resulting in reduced tumor growth. Zika virus preferentially targets cancer stem cells in glioblastoma, resulting in reduced tumor growth.