Haploinsufficient Tumor Suppressor Gene Research Articles

Abstract B042: A screen to identify modifiers of BRCA1 protein level for cancer prevention and treatment

Abstract The breast cancer susceptibility gene 1 (BRCA1) is a nuclear phosphoprotein involved in DNA double-strand break (DSB) repair and maintenance of genome stability. A single BRCA1 mutation leads to reduced levels of functional BRCA1 protein, such that DSB repair is compromised. It has been proposed that BRCA1 is a haploinsufficient tumor suppressor gene in some contexts, which when lost accelerates the mutation rate of other critical genes thus increasing neoplastic transformation. BRCA1 expression levels are modified through exogenous exposures, and modulating BRCA1 expression may alter tumour onset, progression, and treatment options. We hypothesize that compounds that increase BRCA1 expression and function may prevent the outgrowth of BRCA1-associated breast cancer. By contrast, compounds that reduce BRCA1 protein level could be used as combination therapries in conjunction with poly-ADP ribose polymerase (PARP) inhibitors. To achieve these goals, we generated BRCA1-reporter cell lines. Using CRISPR-assisted genome editing, we endogenously tagged the BRCA1 protein with HiBiT, an 11 amino acid luminescent tag. BRCA1-targeting was verified by sequencing and immunoblotting, and functional validation was performed with known modulators of BRCA1 expression. Our tests confirmed that HiBiT-tagging allows for sensitive measurement of BRCA1 protein levels, therefore permitting screenability. BRCA1-reporter cells were subsequently used to conduct a pilot screen (64 epigenetic-modifying drugs) and a high-content screen (6,000 compounds). We identified several compounds that modified BRCA1 protein expression (B-score >3*SD). The epigenetic drug library yielded five repressors and two activators, and the high-content screen identified 163 repressors and 12 activators. Validation studies have confirmed that the activator drug, aloxistatin can elevate BRCA1-HiBiT levels in a dose-dependent manner. Additionally, aloxistatin treatment increased BRCA1 expression and function in a panel of breast cells. We have also assessed 9 down-regulators of BRCA1 from the pilot and high-content screens. Each of these nine compounds reduced the expression of BRCA1 protein and synergized with the PARP inhibitor olaparib to inhibit cancer cell growth. Collectively, our ongoing studies suggest that BRCA1 activation may reduce cancer phenotypes and BRCA1 repression can sensitize breast cancer cells to PARP-inhibition and other chemotherapies. Overall, using our reporter cell lines we have identified a number of unique compounds that effectively modify BRCA1 expression and function that may have utility in the treatment and prevention of BRCA1-associated cancers. Citation Format: Erin Sellars, Margarita Savguira, Joanne Kotsopoulos, Leonardo Salmena. A screen to identify modifiers of BRCA1 protein level for cancer prevention and treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B042.

The function of BCL11B in base excision repair contributes to its dual role as an oncogene and a haplo-insufficient tumor suppressor gene.

Genetic studies in mice and human cancers established BCL11B as a haploinsufficient tumor suppressor gene. Paradoxically, BCL11B is overexpressed in some human cancers where its knockdown is synthetic lethal. We identified the BCL11B protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro DNA repair assays demonstrated that both BCL11B and a small recombinant BCL11B213-560 protein lacking transcription regulation potential can stimulate the enzymatic activities of two base excision repair (BER) enzymes: NTHL1 and Pol β. In cells, BCL11B is rapidly recruited to sites of DNA damage caused by laser microirradiation. BCL11B knockdown delays, whereas ectopic expression of BCL11B213-560 accelerates, the repair of oxidative DNA damage. Inactivation of one BCL11B allele in TK6 lymphoblastoid cells causes an increase in spontaneous and radiation-induced mutation rates. In turn, ectopic expression of BCL11B213-560 cooperates with the RAS oncogene in cell transformation by reducing DNA damage and cellular senescence. These findings indicate that BCL11B functions as a BER accessory factor, safeguarding normal cells from acquiring mutations. Paradoxically, it also enables the survival of cancer cells that would otherwise undergo senescence or apoptosis due to oxidative DNA damage resulting from the elevated production of reactive oxygen species.

Cancer aneuploidies are shaped primarily by effects on tumour fitness.

Aneuploidies-whole-chromosome or whole-arm imbalances-are the most prevalent alteration in cancer genomes1,2. However, it is still debated whether their prevalence is due to selection or ease of generation as passenger events1,2. Here we developed a method, BISCUT, that identifies loci subject to fitness advantages or disadvantages by interrogating length distributions of telomere- or centromere-bounded copy-number events. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage specific. BISCUT identified the helicase-encoding gene WRN as a haploinsufficient tumour-suppressor gene on chromosome 8p, which is supported by several lines of evidence. We also formally quantified the role of selection and mechanical biases in driving aneuploidy, finding that rates of arm-level copy-number alterations are most highly correlated with their effects on cellular fitness1,2. These results provide insight into the driving forces behind aneuploidy and its contribution to tumorigenesis.

The oncogenic and immunological roles of histidine triad nucleotide-binding protein 1 in human cancers and their experimental validation in the MCF-7 cell line.

Histidine triad nucleotide binding protein 1 (HINT1) is a haplo-insufficient tumor suppressor gene that plays a significant role in cell proliferation and survival. However, to date, no systematic pan-cancer analysis has been conducted to explore its function in prognosis, and its oncogenic and immunological roles. We also analyzed the role of HINT1 in breast cancer (BC) progression in vitro. An analysis of the HINT1 expression pattern was performed using the TIMER database. The infiltration of immune cells into several cancer types was also studied using the Xena Shiny tool. To determine the relationship between stemness and the expression of HINT1 mRNA, the Spearman correlation test was used with the SangerBox tool. The correlation between HINT1 and functional states in various cancers was determined from the CancerSEA database. The potential role of HINT1 in BC oncogenesis was also investigated by Western blot and Annexin V/PI assays. The Cancer Genome Atlas pan-cancer data analysis suggested that HINT1 was extensively altered in most tumor tissues but not in most adjacent normal tissues. A high expression of HINT1 was associated with the decreased infiltration of cluster of differentiation (CD)4+ T cells. Importantly, increased HINT1 expression was also associated with a large majority of tumors with high stemness and lower stromal, immune, and estimate scores. Further, the expression of HINT1 was significantly associated with the tumor mutational burden (TMB) and microsatellite instability (MSI) in certain tumor types. Finally, HINT1 overexpression was found to impair BC progression by promoting cell apoptosis. HINT1 upregulation also reduced the expression of microphthalmia transcription factor (MITF) and β-catenin in BC Michigan Cancer Foundation-7 (MCF-7) cells, and the phosphorylation of protein kinase B (p-Akt). The present study showed that HINT1 plays an oncogenic role in various cancers and could also be used as a biomarker for BC.

Higher order genetic interactions switch cancer genes from two-hit to one-hit drivers

The classic two-hit model posits that both alleles of a tumor suppressor gene (TSG) must be inactivated to cause cancer. In contrast, for some oncogenes and haploinsufficient TSGs, a single genetic alteration can suffice to increase tumor fitness. Here, by quantifying the interactions between mutations and copy number alterations (CNAs) across 10,000 tumors, we show that many cancer genes actually switch between acting as one-hit or two-hit drivers. Third order genetic interactions identify the causes of some of these switches in dominance and dosage sensitivity as mutations in other genes in the same biological pathway. The correct genetic model for a gene thus depends on the other mutations in a genome, with a second hit in the same gene or an alteration in a different gene in the same pathway sometimes representing alternative evolutionary paths to cancer.

Functional and Biological Implications of CUX1 Mutations and Deletions in Myeloid Neoplasms

',“ authors equally contributeThe CUX1 gene, located on 7q22.1, has been characterized genetically as a haplo-insufficient tumor suppressor gene. Somatic CUX1 mutations (CUX1MT) and deletions (CUX1DEL) have been reported in myeloid neoplasms.The p200CUX1 is the most abundant isoform, containing 4 DNA-binding domains (CR1, CR2, CR3, and HD) with rapid “on” and “off” DNA-binding kinetics. Depending on promoter context, the p110 isoform can be either a repressor or an activator. We aim to define the clinical and biological/functional relevance of CUX1 lesions in myeloid neoplasms.We first established the clinical prevalence and phenotypic associations of CUX1 . In a cohort of 1480 patients with myeloid neoplasms including, lower-risk MDS (24%, n=359), higher-risk MDS (17%, n=249), pAML (22%, n=322), sAML (14%, n=205), MDS/MPN (15%, n=217) and MPN (9%, n=128), we correlated CUX1 lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and concomitant somatic mutations. We found that somatic CUX1MT and CUX1DEL were detected in 4% and 6% of our cohort, respectively. All CUX1MT were heterozygous except in 6% were homozygous (uniparental disomy; UPD) and 7%, compounded heterozygous fashion. CUX1 is under expressed in 60% of MDS with -7/del(7q) and 70% of AML with -7/del(7q) vs. 18% and 8% of diploid controls (P=.004 and P <.0001,respectively). In addition, 15% of MDS and 5% of AML cases with cytogenetic abnormality other than -7/del(7q) showed low expression of CUX1 . Truncations and missense mutations were present in 25% and 75% of mutant cases, respectively. CUX1MT were more common in MDS (52%) than AML(16%) and MDS/MPN (32%; P=.02), while CUX1DEL more frequently exist in RAEB1/2 and sAML (64%) compared to lower-risk MDS (12%), pAML (12%) and MDS/MPN (14%; P <.0001). Regards concomitant genetic events, CUX1 lesions were significantly associated with TET2, ASXL1 and BCOR (P=.02, P=.004, P=.0009 respectively) compared to CUX1 wild type (WT). In contrast to WT which was mutually exclusive with FLT3 (3%). Clonal architecture analyses showed that 36% of CUX1 mutations were founder events with secondary BCOR (25%) and ASXL1 (17%) , while CUX1MT were subclonal in 64% in which TET2 (22%), BCOR (9%), and SF3B1 ( 9%) served as ancestral events. Prognostic analyses showed that both CUX1MT and CUX1DEL were significantly associated with shorter overall survival compared WT (CUX1MT: 56 vs. 94 mo.; P=.02; CUX1DEL: 46 vs. 94 mo., P=.004) where truncating mutations had a significant impact on worse prognosis than WT (39 v s. 94 mo.; P=.01).CUX1 functions as an auxiliary factor in base excision repair. Specifically, the CUT domains within CUX1 stimulate the enzymatic activities of the 8-oxoguanine DNA Glycosylase-1 (OGG1). We therefore verified whether genetic lesions of CUX1, either CUX1MTor CUX1DEL, delay oxidative DNA damage repair and thereby contribute to leukemogenesis by establishing a clonal mutator phenotype. First, we compared 8-oxoG cleavage activity in extracts from HCC1419 cells harboring CUX1 loss-of-heterozygosity (CUX1LOH), Hs578T cells diploid for CUX1 and Hs578T cells expressing CUX1 shRNA. We observed decreased 8-oxoG cleavage in HCC1419 cells and Hs578T-shCUX1 cells in which CUX1 expression was reduced. Secondly, we obtained bone marrow cells from healthy donors and patients with frameshift CUX1MTor del7q, and performed single cell gel electrophoresis (comet assays) to monitor DNA repair following exposure to H2O2. We found that repair of oxidized bases in genomic DNA was delayed in both samples with heterozygous and homozygous CUX1 inactivating mutations and in samples with CUX1DEL. Having noted that CUX1 lesions cause a defect in BER, we performed whole exome sequencing of CUX1MT (n=3) and WT (n=117). Strikingly, samples with either CUX1MT or low CUX1 expression (n=23) harbored a significantly higher number of mutations compared to WT and high CUX1 expression samples (n=156; P=.03, P=.04).Altogether, our results show that reduced CUX1 expression in myeloid cancer cells is associated both with a decrease in DNA repair efficiency and an increase in somatic mutations. We propose that genetic and epigenetic changes that reduce CUX1 expression cause a DNA repair defect that leads to the accumulation of somatic mutations and ultimately, shorter patient survival. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract 4670: TIP60 regulates alternative splicing of Integrin subunit alpha 6

Abstract Differences in RNA processing, specifically RNA splicing, contribute to enormous diversity that can be generated from the eukaryotic genome. Given the increasing evidence to show that splicing can occur co-transcriptionally, chromatin structure and histone modifications have been reported to affect the choice of a splice site. For instance, HDAC inhibitor treatment has been shown to affect alternative splicing. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. TIP60 is a lysine acetyltransferase that acetylates histone and non-histone proteins. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60's role in modulating alternative splicing. To understand TIP60's role in alternative splicing, RNA sequencing was performed in MCF10A cells that were (a) transiently depleted of TIP60 (b) stably overexpressing siRNA-resistant wild-type TIP60 (MCF10A*WT) and (c) stably overexpressing siRNA-resistant catalytically dead TIP60 (MCF10A*KD). Differential splicing analysis was performed using MISO program. We found 130 genes to be differentially spliced when TIP60 was depleted and 59 genes were differentially spliced in MCF10A*KD compared to MCF10A*WT. For both comparisons, the majority of the differential splicing events were skipped exon events. Upon overlapping the differential skipped exon events, we found significant exon 25 skipping of ITGA6, resulting in an isoform that has a shorter cytoplasmic domain. TIP60 depletion results in an increase in the ITGA6 isoform that lacks exon 25, which has been implicated to promote tumor initiation in breast cancer. To identify the splicing factor that regulates this event, we performed a siRNA screen of over 70 splicing factors and identified ESRP2 to regulate splicing of ITGA6 exon 25. The consequence and biological significance of TIP60-ESRP2 mediated alternative splicing will be discussed at the conference. Citation Format: Shreshtha Sailesh Bhatia, H. Phillip Koeffler, Sudhakar Jha. TIP60 regulates alternative splicing of Integrin subunit alpha 6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4670.

Effects of Beclin 1 overexpression on aggressive phenotypes of colon cancer cells.

Beclin 1 is involved in autophagy, differentiation, apoptosis and cancer progression, and functions as a haploinsufficient tumor suppressor gene. The aim of the present study was to elucidate the function of Beclin 1 in colon cancer. A Beclin 1-expressing plasmid was transfected into HCT-15 and HCT-116 cells, and the phenotypes and associated molecules were determined. Beclin 1 transfectants were subcutaneously injected into nude mice to determine tumor growth, and proliferation and apoptosis levels using Ki-67 immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Beclin 1 overexpression inhibited viability as determined using a Cell Counting Kit-8 assay, inhibited migration and invasion as determined using a wound healing assay or Transwell assay, and lamellipodia formation by filamentous actin staining, induced autophagy as determined using electron microscopy, and light chain 3B (LC-3B) expression, and apoptosis as determined using Annexin V staining in the two cell lines (P<0.05). Beclin 1 induced G2 arrest of HCT-15 transfectants as determined using propidium iodide staining (P<0.05), whereas HCT-116 transfectants were arrested in G1 phase (P<0.05). The two transfectants exhibited increased expression of c-Myc, cyclin D1, β-catenin, insulin-response element 1 and 78 kDa glucose-regulated protein compared with the control and mock cells as determined using the reverse transcription-quantitative polymerase chain reaction (P<0.05). Beclin 1 overexpression upregulated LC-3B and cyclin-dependent kinase 4 expression, but downregulated cyclin E expression of the cancer cell lines as determined using western blot analysis (P<0.05). Beclin 1 expression in vivo significantly suppressed the proliferation of colon cancer cells in xenograft models via inducing apoptosis by TUNEL, and inhibiting proliferation by Ki-67 expression (P<0.05). Beclin 1 overexpression may reverse aggressive phenotypes and suppress colon cancer tumor growth, and be employed as a target molecule for gene therapy of patients with colon cancer.

CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer.

CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/−) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/− MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/− MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.

KMT2D Is a Haploinsufficient Tumor Suppressor in Acute Leukemia

Introduction: Epigenetic dysregulation plays a critical role in hematologic tumorigenesis and cancer progression. Histone-lysine N-methyltransferase 2D (KMT2D), also known as MLL4 (mixed-lineage leukemia 4), belongs to a family of mammalian histone H3 lysine 4 (H3K4) methyltransferases, which is amongst the most frequently mutated epigenetic genes in human cancers. It was reported that deletion of KMT2D inhibits MLL1-fusion-induced acute myeloid leukemia (Santos et al. 2014). However, in human cancers, the vast majority of the mutations in KMT2D is heterozygous deletion. Recent studies have revealed the role of KMT2D in lymphomagenesis, but the potential tumor suppressor function of KMT2D in leukemogenesis remains largely unclear. In addition, KMT2D was found to exist within a macromolecular complex named COMPASS (complex of proteins associated with Set1). KMT2C is another component of the complex, and was validated as another haploinsufficient tumor suppressor gene in acute myeloid leukemia (Chen et al. 2014). Mutations in KMT2D and KMT2C frequently co-occur in hematologic malignancies. Thus, we sought to first examine the role and molecular mechanism of KMT2D in acute leukemia, and then investigate whether co-mutated gene KMT2D and KMT2C have cooperative function in malignancies initiation and progression.Methods: Here we utilized RNAi approach to knockdown the expression of Kmt2d, and an approximate 50% reduction in gene dosage was validated by quantitative RT-PCR. We introduced two independent short-hairpin RNAs (shRNAs) targeting Kmt2d and a shRNA targeting Nf1 into p53 null hematopoietic stem and progenitor cells (hereafter referred to as shKmt2d;shNf1;p53-/- cells), and transplanted them into sublethally irradiated mice to model the impact of haploinsufficient tumor suppressors. The recipient mice were then monitored for the emergence of diseases by complete blood count as well as overall survival. Bone marrow was harvested and analyzed by flow cytometry after sacrifice. Histological analyses of blood smear, liver, spleen and bone marrow were conducted to accurately diagnose the disease. Besides, we established Kmt2d heterozygous and homozygous conditional knockout mouse models (Kmt2d fl/+; Mx1-Cre, Kmt2d fl/fl; Mx1-Cre) to further investigate the respective role of haploinsufficiency and deletion of KMT2D in acute leukemia. To study the cooperative function of KMT2D and KMT2C in leukemogenesis, two genes were co-suppressed by shRNAs in the same aforementioned genetic background (shNf1; p53-/-). Transplantation-based mouse modeling approach was used and the recipient mice were monitored for evidence of hematologic diseases.Results: A significant increase in peripheral white blood cell counts, anemia and thrombocytopenia were noticed in recipients of shKmt2d;shNf1;p53-/- cells over the same period after 4 weeks post-transplantation. Massive hepatomegaly and splenomegaly were displayed in those mice and a dramatic reduction in survival was observed (shKmt2d-1: median survival = 47 days, p = 0.0004 versus shRen; and shKmt2d-2: median survival = 74 days, p = 0.0127 versus shRen). Flow cytometry of cells from bone marrow of sacrificed mice showed a substantial enrichment of doule-positve cells (shRNAs targeting Kmt2d were linked to GFP and those targeting Nf1 were linked to mCherry) as compared to pre-injected, which were filled with leukemic cells that expressed myeloid/lymphoid lineage markers. Pathological analyses demonstrated the onset of acute myeloid/lymphoid leukemia. In Kmt2d and Kmt2c double-knockdown animal assay, the onset of acute myeloid leukemia was promoted compared with single-knockdown groups. (shKmt2d;shKmt2c: median survival = 48.5 days, p < 0.0001 versus shKmt2d; p = 0.0013 versus shKmt2c).Conclusions: KMT2D is a haploinsufficient tumor suppressor in acute leukemia. KMT2D deficiency cooperates with KMT2C to promote the development and progress of acute leukemia. Further understandings of the molecular mechanism of KMT2D as a haploinsufficient tumor suppressor in acute leukemia and the cooperative function of KMT2D and KMT2C in leukemogenesis are demanded, which can provide insights into developing novel therapeutic targets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PAX5-ELN oncoprotein promotes multistep B-cell acute lymphoblastic leukemia in mice

PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (B-ALL) and is involved in various chromosomal translocations that fuse a part of PAX5 with other partners. However, the role of PAX5 fusion proteins in B-ALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human B-ALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in B-ALL development, we generated a knockin mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN-expressing mice efficiently developed B-ALL with an incidence of 80%. Leukemic transformation was associated with recurrent secondary mutations on Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrate that PAX5-ELN affected B-cell development in vitro and in vivo featuring an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Finally, our molecular and computational approaches identified PAX5-ELN-regulated gene candidates that establish the molecular bases of the preleukemic state to drive B-ALL initiation. Hence, our study provides a new in vivo model of human B-ALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development.

Hint1 expression inhibits proliferation and promotes radiosensitivity of human SGC7901 gastric cancer cells.

Gastric cancer is a prevalent, malignant tumor that frequently escapes treatment. Histidine triad nucleotide-binding protein 1 (Hint1) is a haploinsufficient tumor suppressor gene which contributes to intercellular communication, helps to regulate cell proliferation and survival, and is frequently underexpressed in gastric cancer. To examine the involvement of Hint1 in gastric cancer, small interfering RNA was used to knock down Hint1 expression in the human gastric cancer cell line SGC-7901. The data revealed that Hint1 inhibited cell proliferation, reduced radiation-induced DNA damage repair and caused G1 phase arrest, which increased the radiosensitivity of gastric cancer cells. Further mechanistic studies revealed a novel function of Hint1, whereby it acted as a negative regulator of extracellular signal-regulated kinase. These results demonstrated the critical function of Hint1 in the biology of human gastric cancer. Acting as a tumor growth suppressor and a radiosensitive agent, this protein is a potential biomarker and may be an attractive target for specific therapeutic interventions against gastric cancer.

PP2A Inactivation Mediated by PPP2R4 Haploinsufficiency Promotes Cancer Development.

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4 Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825-37. ©2017 AACR.

CTCF genetic alterations in endometrial carcinoma are pro-tumorigenic

CTCF is a haploinsufficient tumour suppressor gene with diverse normal functions in genome structure and gene regulation. However the mechanism by which CTCF haploinsufficiency contributes to cancer development is not well understood. CTCF is frequently mutated in endometrial cancer. Here we show that most CTCF mutations effectively result in CTCF haploinsufficiency through nonsense-mediated decay of mutant transcripts, or loss-of-function missense mutation. Conversely, we identified a recurrent CTCF mutation K365T, which alters a DNA binding residue, and acts as a gain-of-function mutation enhancing cell survival. CTCF genetic deletion occurs predominantly in poor prognosis serous subtype tumours, and this genetic deletion is associated with poor overall survival. In addition, we have shown that CTCF haploinsufficiency also occurs in poor prognosis endometrial clear cell carcinomas and has some association with endometrial cancer relapse and metastasis. Using shRNA targeting CTCF to recapitulate CTCF haploinsufficiency, we have identified a novel role for CTCF in the regulation of cellular polarity of endometrial glandular epithelium. Overall, we have identified two novel pro-tumorigenic roles (promoting cell survival and altering cell polarity) for genetic alterations of CTCF in endometrial cancer.

The CREBBP Acetyltransferase Is a Haploinsufficient Tumor Suppressor in B-cell Lymphoma.

Inactivating mutations of the CREBBP acetyltransferase are highly frequent in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the two most common germinal center (GC)-derived cancers. However, the role of CREBBP inactivation in lymphomagenesis remains unclear. Here, we show that CREBBP regulates enhancer/super-enhancer networks with central roles in GC/post-GC cell fate decisions, including genes involved in signal transduction by the B-cell receptor and CD40 receptor, transcriptional control of GC and plasma cell development, and antigen presentation. Consistently, Crebbp-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of Crebbp was insufficient to initiate malignant transformation, compound Crebbp-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish CREBBP as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.Significance: Loss-of-function mutations of CREBBP are common and early lesions in FL and DLBCL, suggesting a prominent role in lymphoma initiation. Our studies identify the cellular program by which reduced CREBBP dosage facilitates malignant transformation, and have direct implications for targeted lymphoma therapy based on drugs affecting CREBBP-mediated chromatin acetylation. Cancer Discov; 7(3); 322-37. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Hint1 suppresses migration and invasion of hepatocellular carcinoma cells in vitro by modulating girdin activity.

Histidine triad nucleotide-binding protein 1 (Hint1) is a haploinsufficient tumor suppressor gene. Its role in cancer cell migration has not been previously speculated. In the current study, we examined the expression of Hint1 in metastatic and non-metastatic lymph nodes of hepatocellular carcinoma (HCC) patients and further elucidated the effect of Hint1 expression on girdin expression and phosphorylation of AKT and ERK1/2 and on the migration of HCC cells in vitro. Expression of Hint1 and girdin in primary HCC tissues and metastatic and non-metastatic lymph nodes was determined by RT-PCR assays. HepG2 cells were transfected with plasmid vectors overexpressing Hint1 or small interfering RNA (siRNA) targeting Hint1, girdin, Hint1 plus girdin, or the scrambled RNA. Migration and invasion of HCC cells were examined by wound and Transwell assays. Protein expression was detected by immunofluorescence and immunoblotting assays. RT-PCR assays revealed that the messenger RNA (mRNA) transcript levels of Hint1 were markedly lower than those of primary HCC tissues and non-metastatic lymph nodes (P<0.01). By contrast, the mRNA transcript levels of girdin were significantly higher than non-metastatic lymph nodes (P<0.05). Furthermore, siRNA knockdown of HINT1 resulted in a significant increase in the mRNA transcript levels of girdin in HepG2 cells (P<0.05). Wound assays and Transwell assays showed that Hint1 knockdown by siRNA significantly enhanced the migration and invasion of HepG2 cells compared to HepG2 cells transfected with scrambled siRNA. Hint1 knockdown also led to significantly increased phosphorylation of girdin and AKT in HepG2 cells (P<0.05), which, however, was effectively aborted by girdin knockdown by siRNA (P<0.05). Hint1 is downregulated in metastatic lymph nodes and is implicated in migration and invasion of HCC cells in vitro by modulating girdin and AKT expression and phosphorylation. The Hint1-girdin-AKT signaling axis should be further dissected for its role in HCC migration and invasion and may be therapeutically targeted to suppress tumor growth and metastasis.

Haploinsufficiency of the ESCRT Component HD-PTP Predisposes to Cancer.

Endosomal sorting complexes required for transport (ESCRT) drive cell surface receptor degradation resulting in attenuation of oncogenic signaling and pointing to a tumor suppressor function. Here, we show that loss of function of an ESCRT protein (HD-PTP encoded by the PTPN23 gene, located on the tumor suppressor gene cluster 3p21.3) drives tumorigenesis invivo. Indeed, Ptpn23(+/-) loss predisposes mice to sporadic lung adenoma, B cell lymphoma, and promotes Myc-driven lymphoma onset, dissemination, and aggressiveness. Ptpn23(+/-)-derived tumors exhibit an unaltered remaining allele and maintain 50% of HD-PTP expression. Consistent with the role of HD-PTP in attenuation of integrin recycling, cell migration, and invasion, hemizygous Ptpn23(+/-) loss increases integrin β1-dependent B cell lymphoma survival and dissemination. Finally, we reveal frequent PTPN23 deletion and downregulation in human tumors that correlates with poor survival. Altogether, we establish HD-PTP/PTPN23 as a prominent haploinsufficient tumor suppressor gene preventing tumor progression through control of integrin trafficking.

HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis and is down-regulated by both deletion and epigenetic alterations

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1, HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in many human tumors. Here, we report HACE1 as a candidate tumor suppressor gene down-regulated by a combination of deletion and epigenetic mechanisms. HACE1 deletions were observed in 40% of B-cell lymphoma tumors. Hypermethylation of the HACE1 promoter CpG177 island was found in 60% (68/111) of cases and in all tested B-cell lymphoma lines. Using HDAC inhibitors, we observed predominantly inactive chromatin conformation (methylated H3 histones H3K9me2) in HACE1 gene promoter region. We demonstrated in Ramos and Raji cells that down-regulation of HACE1 expression was associated with a significant decrease in apoptosis and an accumulation of cells in the S and G2/M phases. Our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and can be downregulated by deacetylation of its promoter region chromatin, which makes HACE1 a potential target for HDAC inhibitors.

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.

Hepatocellular carcinoma mouse models: Hepatitis B virus-associated hepatocarcinogenesis and haploinsufficient tumor suppressor genes.

The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine.