Shiaw-Yih Lin

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 −/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 −/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 −/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 −/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2s function and as a result leads to infertility and genomic instability in mice.

ZNF668 functions as a tumor suppressor by regulating p53 stability and function in breast cancer

Genome-wide sequencing studies in breast cancer have recently identified frequent mutations in the zinc finger protein 668 (ZNF668), the function of which is undefined. Here, we report that ZNF668 is a nucleolar protein that physically interacts with and regulates p53 and its negative regulator MDM2. Through MDM2 binding, ZNF668 regulated autoubiquitination of MDM2 and its ability to mediate p53 ubiquitination and degradation. ZNF668 deficiency also impaired DNA damage-induced stabilization of p53. RNA interference-mediated knockdown of ZNF668 was sufficient to transform normal mammary epithelial cells. ZNF668 effectively suppressed breast cancer cell proliferation in vitro and tumorigenicity in vivo. Taken together, our studies identify ZNF668 as a novel breast tumor suppressor gene that functions in regulating p53 stability.

Improved prediction of PARP inhibitor response and identification of synergizing agents through use of a novel gene expression signature generation algorithm

Despite rapid advancement in generation of large-scale microarray gene expression datasets, robust multigene expression signatures that are capable of guiding the use of specific therapies have not been routinely implemented into clinical care. We have developed an iterative resampling analysis to predict sensitivity algorithm to generate gene expression sensitivity profiles that predict patient responses to specific therapies. The resultant signatures have a robust capacity to accurately predict drug sensitivity as well as the identification of synergistic combinations. Here, we apply this approach to predict response to PARP inhibitors, and show it can greatly outperforms current clinical biomarkers, including BRCA1/2 mutation status, accurately identifying PARP inhibitor-sensitive cancer cell lines, primary patient-derived tumor cells, and patient-derived xenografts. These signatures were also capable of predicting patient response, as shown by applying a cisplatin sensitivity signature to ovarian cancer patients. We additionally demonstrate how these drug-sensitivity signatures can be applied to identify novel synergizing agents to improve drug efficacy. Tailoring therapeutic interventions to improve patient prognosis is of utmost importance, and our drug sensitivity prediction signatures may prove highly beneficial for patient management.Personalized medicine: Signature-guided cancer therapyPersonalized cancer therapy is one of the holy grails of oncology, as the ability to determine what treatment would best benefit a patient would serve not only to improve outcomes, but also mitigate side effects from less effective treatments. Here, we develop algorithms to predict what patients will respond to a given therapeutic modality, as well as ways to specifically target any observed phenotype, by integrating large scale data sets that profile cancer cell line gene expression and sensitivity to hundreds of drugs. Furthermore, we show how these gene expression signatures can be used to predict novel synergizing agents to further enhance the efficacy of these therapeutics. Taken together, this work stands to advance the era of personalized medicine by enabling precision medicine approaches in the clinic.

Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model

MCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/− mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/−p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. In addition, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end-joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/− MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be used to develop novel targeted cancer therapies in the future.

Identification of MYST3 as a novel epigenetic activator of ER|[alpha]| frequently amplified in breast cancer

Estrogen receptor α (ERα) is a master driver of a vast majority of breast cancers. Breast cancer cells often develop resistance to endocrine therapy via restoration of the ERα activity through survival pathways. Thus identifying the epigenetic activator of ERα that can be targeted to block ERα gene expression is a critical topic of endocrine therapy. Here, integrative genomic analysis identified MYST3 as a potential oncogene target that is frequently amplified in breast cancer. MYST3 is involved in histone acetylation via its histone acetyltransferase domain (HAT) and, as a result, activates gene expression by altering chromatin structure. We found that MYST3 was amplified in 11% and/or overexpressed in 15% of breast tumors, and overexpression of MYST3 correlated with worse clinical outcome in estrogen receptor+ (ER+) breast cancers. Interestingly, MYST3 depletion drastically inhibited proliferation in MYST3-high, ER+ breast cancer cells, but not in benign breast epithelial cells or in MYST3-low breast cancer cells. Importantly, we discovered that knocking down MYST3 resulted in profound reduction of ERα expression, while ectopic expression of MYST3 had the reversed effect. Chromatin immunoprecipitation revealed that MYST3 binds to the proximal promoter region of ERα gene, and inactivating mutations in its HAT domain abolished its ability to regulate ERα, suggesting MYST3 functioning as a histone acetyltransferase that activates ERα promoter. Furthermore, MYST3 inhibition with inducible MYST3 shRNAs potently attenuated breast tumor growth in mice. Together, this study identifies the first histone acetyltransferase that activates ERα expression which may be potentially targeted to block ERα at transcriptional level.

Abstract 2029: Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnMCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here, we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/- mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/−p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. Additionally, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/- MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be utilized to develop novel targeted cancer therapies in the future.nnCitation Format: Yulong Liang, Hong Gao, Shiaw-Yih Lin, John A. Goss, Chunying Du, Kaiyi Li. Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2015-2029

Abstract 406: A potential new mechanism for PTEN to maintain genome stability

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnPurpose: PTEN (phosphatase and tensin homolog) is among the most mutated or lost tumor suppressors. Recent studies revealed that nuclear PTEN represses tumorigenesis by stabilizing the centromere, a highly condensed heterochromatin domain featured with long array of DNA repeats (namely, satellite DNA), repressive histones, such as trimethylated Histone H3 at Lys9 residue (H3K9me3), and heterochromatin proteins. Our studies are aimed to explore the molecular mechanism that PTEN regulates this heterochromatin structure and thus maintaining genome stability.nnMethods and Results: Co-immunoprecipitation (Co-IP) assay showed that PTEN and the heterochromatin proteins directly interact. Chromatin immunoprecipitation (ChIP) assay showed that PTEN is enriched in satellite locus. Depletion of PTEN leads to abnormal heterochromatin structure, indicated by reduced H3K9m3 foci (detected by immunofluorescent staining) and occupancy to satellite locus (ChIP assay), decreased heterochromatin protein levels (Westernblot analysis), and overexpression of satellite transcripts (quantitative RT-PCR). Through a knockdown-and-mutant rescue strategy, we found that the C-terminal of PTEN is essential for its repressive function on satellite transcription. Furthermore, overexpression of the C-terminal truncated PTEN into PTEN-null BT549 cells, as compared to wild-type PTEN overexpression, lost its function to suppress cell growth (MTT assay), cell cycle progression (flow cytometry analysis) and protection from DNA damage (MTT and colony formation assays).nnConclusions: Our finding showed that PTEN guards the genome stability through maintaining the normal structure of heterochromatin domain.nnCitation Format: Lili Gong, Shiaw-Yih Lin. A potential new mechanism for PTEN to maintain genome stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2014-406

Abstract 1573: TUSC4 functions as tumor suppressor by regulating BRCA1 stability and functions

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnExpression of the BRCA1 tumor suppressor is frequently lost in breast cancer and associated with aggressive phenotypes through as yet undetermined mechanisms. In this study, we demonstrated that the tumor suppressor TUSC4 (Tumor Suppressor Candicate 4) physically interacts with multiple DNA damage response proteins and prevent its degradation by E3 ligase ubqutination pathways. Knockdown of TUSC4 enhanced BRCA1 polyubiquitination, leading to BRCA1 protein degradation and significantly reduced the efficacy of homologous recombination repair. Notably, ectopic expression of TUSC4 effectively suppressed multiple breast cancer cells proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, TUSC4 knockdown was sufficient to transform normal mammary epithelial cells and enhance the sensitivity of cells to PARP inhibitors. Microarray analysis of TUSC4 knockdown also indicated critical changes and the global impact to cancer development. Therefore, TUSC4 acts as a bona fide tumor suppressor gene through the mechanism of regulating BRCA1 protein stability and function.nnCitation Format: Yang Peng, Shiaw-Yih Lin. TUSC4 functions as tumor suppressor by regulating BRCA1 stability and functions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2014-1573

Abstract 354: The replication stress response defect is associated with tumor-initiating cell formation

Breast cancer is the most prevalent cancer in women and early diagnosis of breast cancer greatly improves successful treatment and survival rate. Therefore, it is essential to expand current understanding in breast tumorigenesis in order to find specific treatment to target breast cancer in its precancerous or earliest stage. To date, two important observations have been made in terms of precancerous and early breast caner development. First, replication stress response (RSR), also referred to as intra-S checkpoint, serves as a barrier to prevent the occurrence of precancerous lesions and tumorigenesis. When normal cells encounter DNA damages resulting from oncogenic stress, these damages will trigger RSR and provoke cell cycle arrest for repairing damaged DNA or force damaged cells to undergo senescence or apoptosis. However, when RSR is defective, the cells with DNA damage will be able to break the barrier and transform to cancer cells. Second, tumor-initiating cells, which possess stem cell-like properties, are also required for tumorigenesis. Tumor-initiating cells are on the top of the tumor development hierarchy. Therefore, they form in the early stage and then differentiate themselves into nontumorgenic cancer cell to give the heterogeneity of tumor. Even though both processes strongly regulate tumorigenesis, it remains unclear whether defective RSR is associated with tumor-initiating cell formation. To gain insight into breast tumorigenesis from this aspect, RSR-intact cells were given oncogenic stress by exogenous expression of cyclin E, a major oncogene in breast cancer. Once the cells encounter oncogenic-stress-induced DNA damage, several key RSR genes, such as ataxia telangiectasia and Rad3 related (ATR), are then suppressed in order to allow damaged cells to escape from senescence. By measuring the expression changes of transcriptional landscape in RSR-deficient model system, the molecular profile of RSR defective cells are more similar to breast mammary stem cell and breast tumor-initiating cells. Furthermore, the RSR defective cells increase ALDH1, a marker of normal and malignant human mammary stem cell, and increase the ability of mammosphere formation, an indicator of self-renewal property of mammary epithelial stem cells.Importantly, we have also identified EGFR/MEK1 signaling as a key pathway that renders RSR defective cells the ability to escape from senescence or evade apoptosis and acquire tumor-initiating properties. Our findings not only provide insight into the RSR defective function network and the association between RSR defect and tumor-initiating cell formation in early breast cancer development, but also identify the key pathway for breast cancer target therapy and prevention. Citation Format: Curtis Chun-Jen Lin, Hui Dai, Shiaw-Yih Lin. The replication stress response defect is associated with tumor-initiating cell formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 354. doi:10.1158/1538-7445.AM2014-354

Abstract 3342: Characterization and targeting of BRIT1 deficiency in liver cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and there are no safe and effective therapeutic agents yet to treat HCC after diagnosis. However, in HCC, genomic instability is the major remarkable hallmark, especially in the tumors at the late stage and metastatic tumors.nnThus, we wonder if targeting genomic instability can inhibit tumor growth innnHCC. BRIT1/MCPH1 has been identified to be one of the key DNA damage and repair proteins, which harbors three BRCT domains, conserved in BRCA1 and some other proteins involved in DNA damage response pathways. Our previous studies and other reports have illustratednnBRIT1’s essential roles in mitotic and meiotic homologous recombination DNA repair and maintaining genomic stability, which functions through interacting with BRCA2/Rad51 and/or chromatin structure remodeling protein SWI/SNF. Recently, we identified that BRIT1 is aberrantly expressed in ∼25% of HCC samples (n=28).nnIn these samples, the locus of BRIT2 gene showed notable loss of heterogeneity (nearlynn50%). We also found a somatic mutation in BRIT1, which occurred in the splicing donor site of intro 10 in the BRIT1 gene, leading to a truncated protein.nnFunctional analysis showed that this mutation can cause failure of foci formation of BRIT1 under irradiation. Importantly, given that BRIT1-deficient cells exhibited defective DNA repair described above, our in vitro studies showed that the BRIT1-low expressing HCC cells HepG2 and Hep3B are more sensitive to olaparib, the inhibitor of poly(ADP-ribose) polymerase (PARPi), compared to the BRIT1-proficient cell SNU-449, which was determined by WST-1 and colony formation assays.nnFurthermore, we also test the inhibitory effect of olaparib in the xenograft mouse model, and found that PARPi can significantly suppress tumor growth of Hep3B xenografts. Collectively, our results clearly demonstrate thatnnBRIT1 is mutated or aberrantly expressed in HCC, and targeting BRIT1 deficiency by PARP inhibitors may lead to novel and effective targeted therapies to treat BRIT1-deficientnnHCC.nnCitation Format: Yulong Liang, Lihou Yu, Dongxiao Zhang, Hong Gao, Shiaw-Yih Lin, Kaiyi Li. Characterization and targeting of BRIT1 deficiency in liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2013-3342nnNote: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.