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Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line–derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

Supplemental Figures

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PDF file - 4178K, Supplemental Figure 3. Imatinib treatment does not induce a senescence phenotype.

PDF file - 1731K, Supplemental Figure 1. SKP2 expression levels do not predict p27Kip1 levels after imatinib therapy.

PDF file - 3386K, Supplemental Figure 2. GIST cell growth completely recovers after imatinib washout.

PDF file - 1110K, Supplemental Figure 4. Dual siRNA transfection of E2F4/LIN54 leads to an increased apoptotic response after imatinib while single knock-down of DREAM complex subunits does not.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line–derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

Perfectly orchestrated periodic gene expression during cell cycle progression is essential for maintaining genome integrity and ensuring that cell proliferation can be stopped by environmental signals. Genetic and proteomic studies during the past two decades revealed remarkable evolutionary conservation of the key mechanisms that control cell cycle–regulated gene expression, including multisubunit DNA-binding DREAM complexes. DREAM complexes containing a retinoblastoma family member, an E2F transcription factor and its dimerization partner, and five proteins related to products of Caenorhabditis elegans multivulva (Muv) class B genes lin-9, lin-37, lin-52, lin-53, and lin-54 (comprising the MuvB core) have been described in diverse organisms, from worms to humans. This review summarizes the current knowledge of the structure, function, and regulation of DREAM complexes in different organisms, as well as the role of DREAM in human disease.

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.

Triple negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive, and when metastatic is often drug resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for TNBC patients. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models to the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.SignificanceTNBC is a heterogenous disease, often defined by the absence of a therapeutic target. Our recent results show sGAGs may provide a viable biomarker for Triplatin in patients with TNBC, producing a significant advantage over carboplatin in this setting. Selective precision medicine agents, such as Triplatin, that are active against chemotherapy-resistant disease and exploit molecular biomarkers like sGAGs may significantly benefit patients in this setting.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Introduction: Mutant p53 drives several hallmarks of cancer through a gain-of-function oncogenic program. The oncogenicity of mutant p53 includes sustained proliferation, increased survival and resistance to therapy, increased invasion and metastasis, and genomic instability. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. We and others observe that proteasome inhibitors cause paradoxical depletion of mutant p53 in lung cancer cell lines. Objectives: In this project, we sought to determine the mechanism(s) of mutant p53 depletion by proteasome inhibition and its therapeutic implications in lung cancer. Methods: Human lung cancer cell lines bearing mutant p53 and wildtype p53 (WTp53) were used in this study. An isogenic panel of cell lines with mutant p53, WTp53 and a vector control was also used to determine mutant p53 dependency of the observed effects. Western blotting, immunoprecipitation, siRNA technology, DNA transfection were used for protein detection, protein-protein interactions, gene knock-down and overexpression respectively. Cycloheximide chase assay was used to determine protein degradation. The cancer genome atlas (TCGA) analysis was performed using the cBioPortal platform. Results: Proteasome inhibitors resulted in depletion of mutant p53 in the lung cancer cell line H1975. This effect was also confirmed using the isogenic model described above. Depletion of mutant p53 occurred through a degradation mechanism and was time dependent. While WTp53 accumulated at initial timepoints of proteasome inhibition, mutant p53 did not accumulate but instead depleted with time. This suggested that the proteasome is not the primary mechanism for mutant p53, in contrast to WTp53. Since autophagy is an alternative mechanism of protein degradation in eukaryotes, we evaluated for, and found evidence of autophagy upon proteasome inhibition. We further showed that autophagic degradation of mutant p53 could involve the heat shock protein 70. Our TCGA data analysis showed proteasome genes amplification in mutant p53 lung cancers, suggesting a possible functional dependency. Furthermore, RNA sequencing analysis showed upregulation of most proteasome genes upon stable expression of the R273H mutant in p53-null lung cancer cell line. Finally, in vitro cell viability assays using two different proteasome inhibitors showed increased sensitivity of the lung cancer cell line with mutant p53 compared to WTp53. Conclusion: The cumulative evidence of proteasome hyperactivity, degradation of mutant p53 and in vitro evidence of increased susceptibility of mutant p53 lung cancer cell lines suggests that proteasome inhibitors warrant a closer attention for therapeutic targeting of mutant p53 in lung cancer. Citation Format: Eziafa I. Oduah, Larisa Litovchick, Steven R. Grossman. Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 422.

Simple Summary Human papillomaviruses are responsible for around 5% of all cancers, and to date there are no anti-viral therapeutics available for treating these cancers. In this report we demonstrate that in HPV positive cells the transcriptional repressor DREAM complex is disrupted by E7 proteins, with a resulting increase in expression of DREAM target genes. Expression of a mutant DREAM component, LIN52 S20C, competes with E7 and partially rescues DREAM complex formation. This restoration attenuates the growth of HPV positive cells, including HPV positive cervical cancer cell lines. We propose that restoration of the DREAM complex in HPV positive cancers is a novel therapeutic approach that could be adapted to aid in the treatment of these cancers. Abstract High-risk (HR) human papillomaviruses are known causative agents in 5% of human cancers including cervical, ano-genital and head and neck carcinomas. In part, HR-HPV causes cancer by targeting host-cell tumor suppressors including retinoblastoma protein (pRb) and RB-like proteins p107 and p130. HR-HPV E7 uses a LxCxE motif to bind RB proteins, impairing their ability to control cell-cycle dependent transcription. E7 disrupts DREAM (Dimerization partner, RB-like, E2F and MuvB), a transcriptional repressor complex that can include p130 or p107, but not pRb, which regulates genes required for cell cycle progression. However, it is not known whether disruption of DREAM plays a significant role in HPV-driven tumorigenesis. In the DREAM complex, LIN52 is an adaptor that binds directly to p130 via an E7-like LxSxE motif. Replacement of the LxSxE sequence in LIN52 with LxCxE (LIN52-S20C) increases p130 binding and partially restores DREAM assembly in HPV-positive keratinocytes and human cervical cancer cells, inhibiting proliferation. Our findings demonstrate that disruption of the DREAM complex by E7 is an important process promoting cellular proliferation by HR-HPV. Restoration of the DREAM complex in HR-HPV positive cells may therefore have therapeutic benefits in HR-HPV positive cancers.

ContextThe ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited.ObjectiveThis study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype.MethodsPR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established.ResultsSTICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes.ConclusionActivated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.

Summary The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer., Graphical abstract, Highlights • Paf KO suppresses lung adenocarcinoma (LUAD) development • PAF depletion induces cell quiescence and growth arrest of LUAD cells • PAF drives the global expression of cell-cycle genes by remodeling the DREAM complex • Pharmacological mimicking of PAF-depleted transcriptomes inhibits LUAD growth, Bypassing cell quiescence and cell-cycle arrest is a crucial step for tumorigenesis. Here, Kim et al. show that a PAF-remodeled DREAM complex induces the global expression of cell-cycle genes, thereby evading quiescence and promoting lung tumorigenesis.

Detection of the protein antigens in immunoblots prepared with immunoprecipitated protein antigens can be affected by the presence of high amounts of the immunoprecipitating antibodies. When it is not possible to use the immunoprecipitating antibodies and the primary antibodies raised in different species, this protocol provides a convenient and inexpensive alternative to achieve optimal detection of immunoprecipitated protein antigens. In this protocol, a nitrocellulose or polyvinylidene fluoride membrane containing immunoprecipitated protein samples is rinsed with ultrapure H2O after the transfer of proteins and detection of the total proteins using Ponceau S dye (optional). Blocking solution is applied to the membrane, and the membrane is incubated and then rinsed off (optional) before addition of the primary antibody labeled with biotin. After washing, the membrane is incubated with enzyme- or fluorochrome-labeled avidin for detection.

Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane. The protein of interest can be purified or mixed with other proteins as in cell or tissue extracts. Usually immunoblotting combines the resolution of proteins by gel electrophoresis with immunochemical detection and is referred to as “western blotting.” Immunoblotting can be used to determine the presence and the steady-state level of the protein of interest in the sample, its relative molecular weight, and the distribution of the protein between cellular fractions. Immunoblotting can be performed using the antibodies raised against synthetic peptide antigens modified to mimic posttranslational modifications of proteins, such as phosphorylation and acetylation, to study these modifications in the protein of interest in vivo. When antibodies against the protein of interest are not available, immunoblotting can be performed using antibodies that specifically recognize the recombinant epitope tags (hemagglutinin [HA]-, Flag-, cMyc-, or glutathione-S-transferase [GST]) fused to the protein of interest using recombinant DNA techniques. Immunoblotting has a variety of research, clinical, and forensic medicine applications. It is also one of the standard techniques for characterization of antibodies from different samples of polyclonal sera or hybridoma supernatants.

SummaryHumanDYRK1Agene encoding Dual-specificity tyrosine (Y)- Regulated Kinase 1A (DYRK1A) is a dosage-dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in the DNA double-strand break (DSB) repair signaling. This novel function of DYRK1A is mediated in part by its interaction with ubiquitin-binding protein RNF169 that regulates the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair. Accumulation of RNF169 at the DSB sites promotes homologous recombination (HR) by limiting the recruitment of the scaffold protein 53BP1 that promotes NHEJ by protecting the DNA ends from resection. Inducible overexpression of active, but not the kinase inactive, DYRK1A in U-2 OS cells inhibited accumulation of 53BP1 at the DSB sites in RNF169-dependent manner. Mutation of DYRK1A phosphorylation sites in RNF169 or pharmacological inhibition of DYRK1A using harmine decreased the ability of RNF169 to displace 53BP1 from radiation-induced DSB sites. In order to further investigate the role of DYRK1A in regulation of DNA repair, we used CRISPR-Cas9 mediated knockout of DYRK1A in human and mouse cells. Interestingly, knockout of DYRK1A also caused a defect in 53BP1 DSB recruitment that was independent of RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through several mechanisms. U-2 OS cells devoid of DYRK1A displayed an increased DNA repair and HR efficiency, and showed a decreased sensitivity to the PARP inhibitor olaparib when compared to control cells. Given evidence of its altered expression in human cancers, DYRK1A levels could represent a significant determinant of the DNA damaging therapy response.

C-terminal binding protein 2 (CtBP2) is elevated in epithelial ovarian cancer, especially in the aggressive and highly lethal subtype, high-grade serous ovarian cancer (HGSOC). However, whether HGSOC tumor progression is dependent on CtBP2 or its paralog CtBP1, is not well understood. Here we report that CtBP1/2 repress HGSOC cell apoptosis through silencing of death receptors (DRs) 4/5. CtBP1 or 2 knockdown upregulated DR4/5 expression, and triggered autonomous apoptosis via caspase 8 activation, but dependent on cell-type context. Activation of DR4/5 by CtBP1/2 loss also sensitized HGSOC cell susceptibility to the proapoptotic DR4/5 ligand TRAIL. Consistent with its function as transcription corepressor, CtBP1/2 bound to the promoter regions of DR4/5 and repressed DR4/5 expression, presumably through recruitment to a repressive transcription regulatory complex. We also found that CtBP1 and 2 were both required for repression of DR4/5. Collectively, this study identifies CtBP1 and 2 as potent repressors of DR4/5 expression and activity, and supports the targeting of CtBP as a promising therapeutic strategy for HGSOC.

Overexpression of the oncogene MYBL2 (B-Myb) is associated with increased cell proliferation and serves as a marker of poor prognosis in cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adapter for assembly of both the DREAM and MMB complexes, by a mechanism that requires S28 phosphorylation site in LIN52. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification, and establish the rationale for targeting B-Myb to restore cell cycle control.

Vesicle trafficking regulates epithelial cell migration by remodeling matrix adhesions and delivering signaling molecules to the migrating leading edge. Membrane fusion, which is driven by soluble N-ethylmaleimide-sensitive factor associated receptor (SNARE) proteins, is an essential step of vesicle trafficking. Mammalian SNAREs represent a large group of proteins, but few have been implicated in the regulation of cell migration. Ykt6 is a unique SNARE existing in equilibrium between active membrane-bound and inactive cytoplasmic pools, and mediating vesicle trafficking between different intracellular compartments. The biological functions of this protein remain poorly understood. In the present study, we found that Ykt6 acts as a negative regulator of migration and invasion of human prostate epithelial cells. Furthermore, Ykt6 regulates the integrity of epithelial adherens and tight junctions. The observed anti-migratory activity of Ykt6 is mediated by a unique mechanism involving the expressional upregulation of microRNA 145, which selectively decreases the cellular level of Junctional Adhesion Molecule (JAM) A. This decreased JAM-A expression limits the activity of Rap1 and Rac1 small GTPases, thereby attenuating cell spreading and motility. The described novel functions of Ykt6 could be essential for the regulation of epithelial barriers, epithelial repair, and metastatic dissemination of cancer cells.

Detection of the antigen on an immunoblot can be achieved by either enzyme-based detection systems or using detection reagents labeled with fluorochromes. Two major types of enzyme-based detection systems are used in immunoblotting based on either horseradish peroxidase (HRP)–or alkaline phosphatase (AP)–coupled antibodies, and a range of soluble substrates that yield insoluble colored products (chromogenic detection) or generate light (chemiluminescent detection). This protocol describes chromogenic detection with AP as well as both chromogenic and chemiluminescent methods of detection with HRP. Before the detection step, the nitrocellulose or polyvinylidene fluoride (PVDF) membrane is incubated with the antigen using enzyme-conjugated primary antibodies or secondary detection reagents (such as species-specific anti-immunoglobulin antibodies, streptavidin, or Protein A) and washed. An appropriate substrate solution is applied to the membrane, and the signal is detected in the course of the enzymatic reaction, resulting either in development of insoluble colored products (chromogenic substrates) or in generation of light (chemiluminescent substrates). This protocol also describes use of detection reagents labeled with fluorochromes. The nitrocellulose or PVDF membrane incubated with fluorochrome-labeled detection reagents is rinsed with PBS and processed for image capturing with appropriate imaging equipment.

The ability of ovarian steroids to modulate ovarian cancer (OC) risk remains controversial. High grade serous ovarian carcinoma (HGSC), the most aggressive OC subtype, contains abundant estrogen (ER; 76%) and progesterone (PR; 35%) receptors. Progesterone is thought to be a primarily protective factor that reduces OC risk but the relevance of PR actions in HGSC is unknown. Our IHC analyses of human clinical tissues showed robust expression of total and activated phospho-S294 PR within serous tubal intraepithelial carcinomas (STIC), precursor HGSC lesions of fallopian tube epithelia (FTE), suggesting a role for PR signaling in early disease. To explore the molecular mechanisms of PR isoform actions, p53-dominant negative mutant FTE lines expressing the PR-A or PR-B isoform were created. Progestin treatment (R5020 or progesterone) of these models induced spheroid formation and reaggregation, transwell migration and collagen invasion. RNAseq analyses revealed enrichment of cell cycle progression pathways; in particular, the gene targets of the repressive DREAM complex that drives quiescent cell fate. Progestins induced inhibition of proliferation (decreased Ki67; decreased BrdU; increased B-galactosidase) and accumulation of cells in G0/G1 that was reversed following growth factor stimulation. Subsequent immunoprecipitation of DREAM (G0) and B-MYB/MMB complexes (G1/S) confirmed that PR-driven changes in cell fate occur via regulation of the DREAM complex. PR activation enhanced DREAM and suppressed B-MYB/MMB complex formation. PR also induced transcriptional regulation of DREAM/B-MYB/MMB genes (LIN9, MYBL2, DYRK1A, FOXM1) via promoter recruitment and mRNA regulation. Interestingly, DREAM complexes contained PR and PR was co-recruited to DREAM binding sites within DREAM target promoters, along with the DREAM complex proteins p130 and E2F4. The relevance of the quiescent state to early OC phenotypes was examined through the disruption of DREAM/DYRK1s by pharmacological inhibition (harmine) , HPV E6/E7 expression or the depletion of DYRK1A/B kinases that promote DREAM complex formation. These manipulations blocked progestin-induced cell cycle arrest and attenuated PR-driven gene expression and associated OC phenotypes. Our results reveal that activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Taken together, our data suggest an alternative perspective on the tenant that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease. Our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting activated PRs, DREAM complexes, and/or DYRKs (Supported by the Minnesota Ovarian Cancer Alliance to LJM and CAL). Citation Format: Laura J. Mauro, Megan I. Seibel, Caroline H. Diep, Angela Spartz, Carlos Perez Kerkvliet, Hari Singhal, Elizabeth M. Swisher, Lauren E. Schwartz, Ronny Drapkin, Siddharth Saini, Fatmata Seesay, Larisa Litovchick, Carol A. Lange. Progesterone receptors promote quiescence & ovarian cancer cell phenotypes via modulation of DREAM in p53-mutant fallopian tube models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 744.

Immunoprecipitated proteins can be readily analyzed by immunoblotting. Proteins can be efficiently eluted from the Protein A or similar beads by addition of the SDS-PAGE sample loading buffer and heating at 95°C. This elution procedure will also remove the capturing antibody from the beads unless the antibody was cross-linked to the beads. Alternatively, the immunoprecipitated proteins as well as non-cross-linked capture antibodies can be eluted from the beads using low (2.1–2.8) or high (10–11) pH conditions. Incubation of the immunoprecipitates with the excess of the competing peptide allows the elution of the captured proteins without contamination of the sample with the antibodies present in the immunoprecipitates. However, this option is not always available, and the cost of competing peptide can be prohibitive for the routine immunoprecipitation/immunoblotting experiments. In this protocol, elution of the immunoprecipitated proteins from the beads is performed by mixing Protein A or similar beads containing the immunoprecipitated protein antigens of interest with SDS-PAGE sample buffer and boiling to prepare samples for protein gel electrophoresis.

For most immunoblots developed with chemiluminescence or with fluorochrome-based detection systems, it is possible to remove the primary and secondary antibodies from the membrane without affecting the bound antigen. This allows you to reuse the membrane for detection of another protein antigen. The blots developed with chromogenic substrates can also be stripped of antibodies and reprobed, but the bands detected in the first round of immunoblotting will remain unaffected. Stripping and reprobing of the membrane are particularly useful when the amount of sample is limited or when it is important to accurately compare the signal between two different protein antigens in the same sample. Examples of such experiments include determining the levels of a protein antigen in a series of samples relative to the loading control and comparison of the phosphorylated form to the total levels of the protein in the sample.

Before probing blots for the presence of an antigen, the total composition of the transferred proteins can be determined by staining the nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Staining for proteins is useful to determine the position of the non-prestained molecular weight markers or individual lanes on the gel and to ensure that efficient transfer has occurred. It can be also used to verify equal loading of the samples in the gel when a comparison of the protein of interest between the different samples is important. The conventional procedures such as Coomassie Blue and silver staining methods used for staining polyacrylamide gels are incompatible with immunoblotting. Ponceau S is the more common staining method in immunoblotting protocols because it is compatible with antibody–antigen binding, is cost efficient, and provides a good contrast between the stained bands and background. In this protocol, nitrocellulose or PVDF membrane is rinsed with ultrapure H2O after the transfer of proteins. Ponceau S dye is applied as an acidic aqueous solution, and the proteins on the membrane are stained with red color. The membrane is briefly destained with water and can be photographed or scanned to obtain the image of the total protein staining. Individual lane positions or the molecular weight standards can be marked with a pencil, if required.

The MuvB transcriptional regulatory complex, which controls cell-cycle-dependent gene expression, cooperates with B-Myb to activate genes required for the G2 and M phases of the cell cycle. We have identified the domain in B-Myb that is essential for the assembly of the Myb–MuvB (MMB) complex. We determined a crystal structure that reveals how this B-Myb domain binds MuvB through the adaptor protein LIN52 and the scaffold protein LIN9. The structure and biochemical analysis provide an understanding of how oncogenic B-Myb is recruited to regulate genes required for cell-cycle progression, and the MMB interface presents a potential therapeutic target to inhibit cancer cell proliferation.

Most protein solutions can be mixed with sample buffer and analyzed directly by immunoblotting. Any protein extraction buffer or solvent that is compatible with gel electrophoresis can be used for preparing samples for immunoblotting. Examples of the protein solutions suitable for immunoblotting include purified proteins, detergent lysates of cells, samples prepared by subcellular fractionation, and protein eluates from chromatography columns. The protein of interest must be sufficiently abundant in the sample to allow for efficient detection, but the total amount of protein in the sample should not exceed the loading capacity of the gel. For this reason, protein antigens of very low abundance must be partially purified from the protein mixtures containing high concentrations of the contaminating proteins. In this protocol, solutions containing the protein antigens of interest are mixed with SDS-PAGE sample buffer and boiled to prepare samples for protein gel electrophoresis.

In this protocol, adherent cells are grown in tissue culture dishes until ready to be used for protein sample preparation. To analyze proteins by immunoblotting, it is necessary to bring the proteins from the sample into soluble form using the buffers, salts, and detergents compatible with the gel electrophoresis procedure. If a protein sample is being prepared for immunoblotting only, the proteins can be solubilized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, as described in this protocol, which ensures rapid and efficient extraction of most cellular proteins because of the presence of SDS, a strong anionic detergent. SDS-PAGE sample loading buffer denatures proteins and disrupts the bonds between the proteins that interact with each other. Therefore, this lysis technique can be detrimental to the applications when the native structure of the protein is essential, including enzymatic activity assays and analysis of protein–protein interactions.

Cancer cells are characterized by uncontrolled proliferation, whereas the ability to enter quiescence or dormancy is important for cancer cell survival and disease recurrence. Therefore, understanding the mechanisms regulating cell cycle progression and exit is essential for improving patient outcomes. The MuvB complex of five proteins (LIN9, LIN37, LIN52, RBBP4, and LIN54), also known as LINC (LIN complex), is important for coordinated cell cycle gene expression. By participating in the formation of three distinct transcriptional regulatory complexes, including DREAM (DP, RB-like, E2F, and MuvB), MMB (Myb-MuvB), and FoxM1-MuvB, MuvB represents a unique regulator mediating either transcriptional activation (during S-G2 phases) or repression (during quiescence). With no known enzymatic activities in any of the MuvB-associated complexes, studies have focused on the therapeutic potential of protein kinases responsible for initiating DREAM assembly or downstream enzymatic targets of MMB. Furthermore, the mechanisms governing the formation and activity of each complex (DREAM, MMB, or FoxM1-MuvB) may have important consequences for therapeutic response. The MMB complex is associated with prognostic markers of aggressiveness in several cancers, whereas the DREAM complex is tied to disease recurrence through its role in maintaining quiescence. Here, we review recent developments in our understanding of MuvB function in the context of cancer. We specifically highlight the rationale for additional investigation of MuvB in high-grade serous ovarian cancer and the need for further translational research.

© 2015 Guiley et al. The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52–p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights fromthe structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.

The oncogeneMYBL2(encoding B-Myb) is a poor prognostic biomarker in many cancers. B-Myb interacts with the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex and promotes expression of late cell cycle genes necessary for progression through mitosis. BothMYBL2amplification and over-expression are associated with deregulation of the cell cycle and increased cell proliferation. Alternatively, by interacting with E2F4-DP1 and p130 or p107, the MuvB core becomes part of the DREAM complex (DP, RB-like, E2F, and MuvB). The DREAM complex opposes MMB by globally repressing cell cycle genes in G0/G1, maintaining the cell in a quiescent state. However, the specific mechanism by which B-Myb alters the cell cycle is not well understood. Our analysis of The Cancer Genome Atlas data revealed significant upregulation of DREAM and MMB target genes in breast and ovarian cancer withMYBL2gain. Given that most of the DREAM target genes are not directly regulated by B-Myb, we investigated the effects of B-Myb on DREAM formation. We found that depletion of B-Myb results in increased DREAM formation in human cancer cells, while its overexpression inhibits DREAM formation in the non-transformed cells. Since the MuvB core subunit LIN52 is essential for assembly of both the DREAM and MMB complexes, we tested whether B-Myb disrupts DREAM by sequestering LIN52. Overexpression of LIN52 did not increase either DREAM or MMB formation, but instead increased the turnover rate of the endogenous LIN52 protein. Interestingly, co-expression of B-Myb increased the expression of both endogenous and overexpressed LIN52 while knockdown of B-Myb had an opposite effect. We found that regulation of LIN52 occurs at the protein level, and that activity of DYRK1A kinase, the enzyme that triggers DREAM complex formation by phosphorylating LIN52, is required for this regulation. These findings are the first to implicate B-Myb in the disassembly of the DREAM complex and offer insight into the underlying mechanisms of poor prognostic value ofMYBL2amplification in cancer. We conclude that B-Myb mediates its oncogenic effects not only by increasing mitotic gene expression by the MMB complex, but also by broad disruption of cell cycle gene regulatory programs through compromised DREAM formation.

Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point, R. Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase, PP2A, results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. Following PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling-modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and, thereby, achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with 75% of women presenting with metastatic disease at the time of diagnosis. High Grade Serous Ovarian Carcinoma (HGSOC) is the most common and aggressive type of epithelial ovarian cancer, with 5-year survival rate of 30% for most patients. Developing of accurate experimental models of HGSOC is necessary to elucidate the disease pathogenesis and to evaluate new treatments. Our study is aimed at determining the roles of the Large Tumor Suppressor 1 and 2 kinases (LATS1/2), and their downstream effector Dual-specificity tyrosine(Y)-Regulated Protein Kinase 1A (DYRK1A), in HGSOC pathogenesis and treatment sensitivity. The Cancer Genome Atlas study revealed copy number losses of the LATS1, LATS2 and DYRK1A genes in 65%, 59% and 38% of HGSOC cases, respectively. Previous studies found that LATS1/2 and DYRK1A mediate a crosstalk between the Hippo tumor suppressor pathway and the G0/G1 cell cycle checkpoint. However, the role of these kinases in HGSOC pathogenesis is not known. We found that loss of both LATS1 and LATS2, or DYRK1A results in increased proliferation of SKOV3 ovarian cancer cells in 2D or 3D cultures, mediated in part by disruption of the DREAM complex that represses cell cycle-regulated genes. Interestingly, we observed upregulation of cyclin D1 and increased phosphorylation of retinoblastoma (RB) family proteins in SKOV3 cells depleted of LATS1/2 or DYRK1A kinases, indicative of aberrant activation of CDK4/6. Similar changes were observed upon depletion of LATS1/2 or DYRK1A using non-transformed human fallopian epithelial cells, suggesting that genetic losses of these kinases could contribute to the HGSOC pathogenesis. Furthermore, we tested whether an aberrant activation of CDK4/6 in SKOV3 cells with low levels of LATS1/2 or DYRK1A could increase their sensitivity to pharmacological CDK4/6 inhibitors such as palbociclib. Using metabolic cell proliferation assays, we found that loss of LATS1/2 or DYRK1A resulted in a significant decrease of palbociclib IC50 in SKOV3 cells. Interestingly, palbociclib treatment increased the DREAM assembly in these cells, resulting in downregulation of DREAM-regulated cell cycle factors such as cyclin A or B-Myb. Our results show that loss of LATS1/2 or DYRK1A could contribute to HGSOC pathogenesis by CDK4/6-mediated disruption of RB family function. Further studies are underway to validate our findings in vivo using orthotopic ovarian tumor xenografts in mice and a bioluminescence imaging approach. In addition, these models will be used for validation of novel therapeutic approaches for HGSOC, such as palbociclib, as well as for the mechanistic studies of the EOC pathogenesis. Citation Format: Fatmata Sesay, Siddharth Saini, Larisa Litovchick. ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-053.

INTRODUCTION: Cell cycle control is an important determinant of cancer progression and treatment response. Two key transcriptional regulatory complexes, DREAM and MMB, ensure coordinated cell cycle dependent gene expression. Though these complexes contain the same protein core called MuvB, they have opposing functions. The DREAM (DP, RB-like, E2F, and MuvB) complex represses over 800 cell cycle genes in G0/G1 and the MMB (Myb-MuvB) complex promotes mitotic gene expression. High expression of B-Myb, an oncogenic transcription factor involved in the MMB complex, is associated with cell cycle deregulation and poor prognosis in several cancers, including ovarian cancer. High B-Myb expression disrupts DREAM formation in human cell lines, resulting in increased proliferation. Previous analysis of TCGA data showed that MYBL2 (encoding B-Myb) undergoes gene copy number gain in 55% of high grade serous ovarian cancer (HGSOC) tumor samples and is associated with poor overall survival. We sought to validate these findings with clinical specimens and to investigate the role of DREAM- and MMB-regulated gene expression in HGSOC patient outcomes. METHODS: We used expression levels of DREAM-and MMB-controlled genes as a functional readout for the status of these opposing transcriptional regulators. This retrospective study utilized tissue bank surgical pathology and cytology samples taken from 52 HGSOC lesions. RT-qPCR gene expression analysis was correlated to clinical and pathologic findings. Demographic information, follow-up, treatment, and outcomes data (age, Stage, optimal debulking, platinum sensitivity, survival) was obtained by chart review. Analyses of TCGA datasets were conducted in parallel. RESULTS: RT-qPCR analysis of DREAM target genes (AURKA, KIF23, CCNB2, and FOXM1) revealed positive and significant correlations between MYBL2 and all genes tested: AURKA (ρ=0.4114, p CONCLUSIONS: Increased expression of selected cell cycle genes correlates to increased formation of MMB, and reduced DREAM assembly in HGSOC tissue. High expression of MYBL2 is associated with deregulated cell cycle gene expression programs in HGSOC. Larger scale studies would clarify the clinical prognostic value of the DREAM- and MMB-regulated gene expression. Citation Format: Audra N. Iness, Lisa Rubinsak, Jessica Chaoul, Mikhail Dozmorov, Cora Uram-Tuculescu, Larisa Litovchick, Sarah Temkin. CLINICAL PATHOLOGIC EXPRESSION OF CELL CYCLE REGULATORY COMPLEXES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-031.

In this protocol, immunoblots are prepared for detection of the proteins of interest by incubation with blocking solution to prevent nonspecific antibody binding, followed by incubation with primary and secondary antibodies. When labeled primary antibody is used, incubation with secondary antibodies can be omitted.

Recently, clinical development of PARP inhibitors (PARPi) expanded from using them as a single agent to combining them with DNA-damaging therapy to derive additional therapeutic benefit from stimulated DNA damage. Furthermore, inhibiting PARP in cancers with BRCA1/2 mutations has been shown to be an effective synthetic lethality approach either as a single agent or in combination with the different DNA damaging agents: chemotherapy or ionizing radiation (IR). However, inherited BRCA1/2 mutations account only for 5–10% of breast cancers, 10–15% of ovarian cancers, and lesser for the other cancers. Hence, for most of the cancer patients with BRCA1/2-proficient tumors, sensitization to DNA-damaging agents with PARPi is significantly less effective. We recently demonstrated that moderate, non-toxic concentrations of NO-donors inhibited BRCA1 expression, with subsequent inhibition of error-free HRR and increase of error-prone non-homologous end joining (NHEJ). We also demonstrated that the effect of NO-dependent block of BRCA1 expression can only be achieved in the presence of oxidative stress, a condition that characterizes the tumor microenvironment and is also a potential effect of IR. Hence, NO-donors in combination with PARPi, with effects limited by tumor microenvironment and irradiated area, suggest a precise tumor-targeted approach for radio-sensitization of BRCA1/2-proficient tumors. The combination with NO-donors allows PARPi to be successfully applied to a wider variety of tumors. The present work demonstrates a new drug combination (NO-donors and PARP-inhibitors) which demonstrated a high potency in sensitization of wide variety of tumors to ionizing radiation treatment., Graphical abstract Image 1

SUMMARY The control of p53 protein stability is critical to its tumor suppressor functions. The CREB binding protein (CBP) transcriptional co-activator co-operates with MDM2 to maintain normally low physiological p53 levels in cells via exclusively cytoplasmic E4 polyubiquitination activity. Using mass spectrometry to identify nuclear and cytoplasmic CBP-interacting proteins that regulate compartmentalized CBP E4 activity, we identified deleted in breast cancer 1 (DBC1) as a stoichiometric CBP-interacting protein that negatively regulates CBP-dependent p53 polyubiquitination, stabilizes p53, and augments p53-dependent apoptosis. TCGA analysis demonstrated that solid tumors often retain wild-type p53 alleles in conjunction with DBC1 loss, supporting the hypothesis that DBC1 is selected for disruption during carcinogenesis as a surrogate for p53 functional loss. Because DBC1 maintains p53 stability in the nucleus, where p53 exerts its tumor-suppressive transcriptional function, replacement of DBC1 functionality in DBC1-deleted tumors might enhance p53 function and chemosensitivity for therapeutic benefit., Graphical Abstract, In Brief Akande et al. describe DBC1 as a negative regulator of CBP p53-directed ubiquitin ligase activity that maintains nuclear p53 stability. DBC1 loss may be selected in tumors to inactivate the p53 pathway. Because expression of DBC1 enhances p53-dependent apoptosis, restoration of DBC1 in null tumors may be of therapeutic value.

The MuvB complex recruits transcription factors to activate or repress genes with cell cycle-dependent expression patterns. MuvB contains the DNA-binding protein LIN54, which directs the complex to promoter cell cycle genes homology region (CHR) elements. Here we characterize the DNA-binding properties of LIN54 and describe the structural basis for recognition of a CHR sequence. We biochemically define the CHR consensus as TTYRAA and determine that two tandem cysteine rich regions are required for high-affinity DNA association. A crystal structure of the LIN54 DNA-binding domain in complex with a CHR sequence reveals that sequence specificity is conferred by two tyrosine residues, which insert into the minor groove of the DNA duplex. We demonstrate that this unique tyrosine-mediated DNA binding is necessary for MuvB recruitment to target promoters. Our results suggest a model in which MuvB binds near transcription start sites and plays a role in positioning downstream nucleosomes., The MuvB complex, which regulates cell cycle dependent gene expression, binds promoter cell cycle genes homology region (CHR) elements. Here the authors solve the structure of LIN54, a component of MuvB, bound to DNA and use it to explain the recognition of a CHR sequence.

E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their ‘core’ cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer., Author Summary The proliferation of mammalian cells is driven by proteins called cyclins, which bind and activate their catalytic partners, the cyclin-dependent kinases (Cdks). Cyclin-Cdk complexes drive cell proliferation by phosphorylating several cellular proteins. This study focuses on the E-type cyclins (cyclins E1 and E2). These proteins were shown to play important roles in cell cycle progression, and are often overexpressed in human cancers. It remained unclear, however, whether in addition to their well-established cell cycle roles E-type cyclins perform distinct tissue-specific functions in vivo, in the living mouse. To address this question we developed a system, which combines gene targeting in embryonic stem cells with high-throughput mass spectrometry. This approach allowed us to determine the identity of cyclin E protein partners, as well as phosphorylation substrates of cyclin E and its associated kinase, Cdk2, in different mouse organs. These analyses identified several novel, previously unanticipated interactors and phosphorylation substrates of cyclin E-Cdk2, and revealed that cyclin E plays distinct molecular functions in different compartments in vivo. This approach can be used to study the in vivo function of essentially any protein, in any model organism, and in any physiological and pathological state.

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.

Electrophoretic transfer of proteins from gels to membranes can be achieved either by complete immersion of a gel–membrane sandwich in a buffer (wet transfer) or by placing the gel–membrane sandwich between absorbent paper soaked in transfer buffer (semidry transfer). For the wet transfer, the sandwich is placed in a buffer tank with platinum wire electrodes. For the semidry transfer, the gel–membrane sandwich is placed between carbon plate or stainless steel electrodes. Both methods are described here and generally work well, although semidry transfer is quicker and often more complete.

This protocol describes Tris/glycine SDS–polyacrylamide gel electrophoresis, also known as SDS discontinuous gel electrophoresis or the Laemmli electrophoresis system. The gel-casting unit is assembled and tested to make sure that there are no leaks. Ammonium persulfate and tetramethylethylenediamine are added to the separating monomer solution, and the bottom (separating) gel is poured and allowed to polymerize. The top (stacking) gel is poured, and the comb is inserted to make the wells. The stacking gel is allowed to polymerize and the comb is then removed. The gel cassette is connected to the buffer chambers and the samples are loaded into the wells. When the electric current is applied, the proteins migrate through the gel with a rate that is roughly proportional to the length of their polypeptide chains. The electrophoresis is stopped when the loading dye reaches the bottom of the separating gel. The gel cassette is disassembled, and the proteins are ready for transfer from the gel onto the membrane.

High-grade ovarian carcinoma (HGSOC) is the most lethal gynecologic cancer with little improvement in survival over the last decade. Identification of the “druggable” cancer-driving pathways could facilitate the development of effective treatments for HGSOC patients. Oncogenomic analyses of HGSOC tumors revealed widespread genome alterations but only a few recurrent mutations that could be targeted by either approved or experimental therapeutics. We observed copy number losses of genes encoding LATS1 and LATS2 tumor suppressor protein kinases in approximately 60% of HGSOC tumors analyzed by TCGA study. Loss of LATS kinases could contribute to cancer pathogenesis because of their important role in the Hippo tumor suppressor pathway that is known to control cell invasion, migration, and proliferation though inhibition of the oncogenic transcription factors YAP and TAZ. Here, we describe an alternative mechanism by which LATS kinases can control cell proliferation and discuss the implications of this finding for HGSOC treatment. In previous studies, LATS2 kinase was shown to promote growth suppressor function of retinoblastoma family. LATS2 phosphorylation of DYRK1A protein kinase promoted assembly of the transcriptional repressor DREAM. DREAM assembles during G0/G1 when RB-like p130 binds E2F4, DP1 and a stable complex of five proteins, including RBBP4, LIN9, LIN37, LIN52, and LIN54 (MuvB core), and inhibits the expression of more than 800 cell cycle-regulated genes. Here, we report that LATS1 and LATS2 function redundantly in the regulation of DREAM in SKOV3 ovarian cancer cells and in the nontransformed fallopian tube epithelial (FTE) cells. Downregulation of LATS1 and LATS2 expression resulted in increased proliferation and promoted transformed phenotype of these cells, suggesting that loss of these genes could contribute to ovarian cancer aggressiveness. Interestingly, we observed that depletion of both LATS kinases in these cells resulted in markedly increased phosphorylation of the retinoblastoma family proteins pRB and p130. This phosphorylation was likely mediated by CDK4/6 kinases because it was completely abolished by treatment of cells with the highly selective CDK4/6 inhibitor, PD-0332991 (palbociclib). We investigated the mechanism of CDK4/6 activation, and found that downregulation of LATS kinases in SKOV3 or FTE cells resulted in a markedly increased expression of cyclin D1 at the protein, but not at the mRNA level. In a recent study, loss of the CDK4/6 inhibitor, p16, correlated with increased sensitivity of ovarian cancer cell lines to palbociclib. Therefore, we tested whether aberrant activation of CDK4/6 in LATS1/2-depleted cells could similarly predispose ovarian cancer cells to palbociclib growth inhibition. Indeed, downregulation of LATS kinases in SKOV3 cells resulted in 3-fold decrease of palbociclib IC50 compared to control cell lines. Importantly, clinical trials using palbociclib and analogous drugs in several types of cancer revealed a wide range of outcomes, emphasizing significance of research that will help to predict tumor sensitivity. Currently, there are no HGSOC-specific clinical trials using palbociclib. Our findings reported here support the concept that frequent genetic losses of LATS1 and LATS2 kinases in HGSOC could provide a rationale for using CDK4/6 inhibitors, such as palbociclib, for treatment of patients suffering from this disease. Citation Format: Siddharth Saini, Fatmata Sesay, Larisa Litovchick. The role of LATS kinases in regulation of CDK4/6 in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B49.

The oncogene MYBL2 (encoding B-Myb) is a poor prognostic biomarker in many cancers. B-Myb interacts with the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex and promotes expression of late cell cycle genes necessary for progression through mitosis. Both MYBL2 amplification and over-expression are associated with deregulation of the cell cycle and increased cell proliferation. Alternatively, by interacting with E2F4-DP1 and p130/p107, the MuvB core becomes part of the DREAM complex (DP, RB-like, E2F, and MuvB). The DREAM complex opposes MMB by globally repressing cell cycle genes in G0/G1, maintaining the cell in a quiescent state. However, the specific mechanism by which B-Myb alters the cell cycle is not well understood. Herein, we show that B-Myb disrupts the DREAM complex by sequestration and stabilization of LIN52. Analysis of The Cancer Genome Atlas data revealed significant upregulation of DREAM and MMB target genes in breast and ovarian cancer with MYBL2 gain. Given that most of the DREAM target genes are not directly regulated by B-Myb, we investigated the effects of B-Myb on DREAM formation. We found that depletion of B-Myb results in increased DREAM formation in cancer cell lines, while its overexpression inhibits DREAM formation in non-transformed cells. Since the MuvB core subunit LIN52 is essential for assembly of both the DREAM and MMB complexes, we tested whether B-Myb disrupts DREAM by sequestering LIN52. Overexpression of LIN52 did not increase either DREAM or MMB formation, but instead increased the turnover rate of the endogenous LIN52 protein. Interestingly, co-expression of B-Myb increased the expression of both endogenous and overexpressed LIN52 while knockdown of B-Myb had an opposite effect. We found that regulation of LIN52 occurs at the protein level, and that activity of DYRK1A kinase, the enzyme that triggers DREAM complex formation by phosphorylating LIN52, is required for this regulation. These findings are the first to implicate B-Myb in the disassembly of the DREAM complex and offer further mechanistic insights for cancers with MYBL2 amplification. We conclude that B-Myb's oncogenic effects are not only secondary to increased mitotic gene expression by the MMB complex, but also broad disruption of cell cycle gene regulatory programs through compromised DREAM formation. Citation Format: Audra N. Iness, Varsha Ananthapadmanabhan, Fatmata Sesay, Mikhail Dozmorov, Larisa Litovchick. The cell cycle gene regulatory DREAM complex is disrupted by oncogenic B-Myb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4288.

Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit.

OS-9, a protein previously uncharacterized, was shown to interact specifically with the intracellular region of the membrane proteinase meprin beta found in brush border membranes of kidney and small intestine. We have shown previously that this cytoplasmic region is indispensable for the maturation of meprin beta, which included an endoplasmic reticulum (ER)-to-Golgi translocation. We characterized OS-9 and found that it is associated with ER membranes and that it is exposed to the cytoplasm. Consistent with the kinetics of maturation of meprin beta, OS-9 associates with meprin beta only transiently, coinciding with ER-to-Golgi transport of meprin beta. The OS-9-binding site in the cytoplasmic domain of meprin beta overlaps the region essential for this transport. We characterized alternatively spliced forms of rat and mouse OS-9, and we found that only the non-spliced form of OS-9 binds to meprin beta, implicating the spliced out segment in the binding, and suggesting the possible mechanism of the regulation of OS-9 function. Taken together, our results indicated that OS-9 may be involved in the ER-to-Golgi transport of meprin beta. Ubiquitous expression of OS-9 raises the possibility that it may interact with other membrane proteins that possess the cytoplasmic moiety homologous to that of meprin beta during their ER-to-Golgi transition.

The retinoblastoma-related pocket proteins pRb, p107, and p130 are implicated in the control of cell proliferation, differentiation, and transformation. The function of pocket proteins is in part mediated by their ability to inhibit specific E2F transcription factors. The transcriptional activity of E2Fs is controlled by alteration of their nucleocytoplasmic localization during the cell cycle. p130 was observed to shuttle between the nucleus and cytoplasm in a heterokaryon fusion assay, suggesting the presence of nuclear and cytoplasmic localization signals. Two independent nuclear localization signals (NLS) that could target reporter proteins to the nucleus in transient transfection and microinjection experiments were identified in the C terminus of p130. In addition to the C-terminal NLS, the intact pocket domain of p130 itself was sufficient for nuclear translocation. Moreover, an additional functional NLS was mapped within the unique Loop region of p130. An N-terminal domain that conferred cytoplasmic localization was identified. Removal of the entire N terminus did not affect the ability of p130 to interact with E2F and to induce growth arrest. A model suggesting that the activity of pRb family members can be regulated by intracellular trafficking of the proteins is proposed.

Human DYRK1A protein kinase is encoded by dosage-dependent gene since an extra copy contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe developmental defects. Current knowledge of DYRK1A’s role in phosphorylation of proteins involved in cell cycle control, transcription and tumor suppression does not fully explain dosage-dependent function of this important kinase. Using MudPIT proteomic analysis, we identified DYRK1A interacting proteins in human T98G cell line. Four independent DYRK1A-HA pull-down samples were analyzed to identify 50 proteins that were specifically detected in 3 out of 4 replicates. This analysis identified WD-repeat protein DCAF7 as major partner of DYRK1A that was most highly enriched among DYRK1A-binding proteins. Furthermore, glycerol gradient ultracentrifugation showed almost exact co-fractionation of DCAF7 and DYRK1A. DCAF7 has been shown to bind several protein kinases including DYRK1A, DYRK1B, HIPK2 and MAP3K1 suggesting that it serves as a scaffold protein in signal transduction. However, the function of DCAF7 is not established yet. In order to understand the functional relationship between DCAF7 and DYRK1A, we undertook MudPIT proteomic analysis of DCAF7 binding proteins in T98G cells and compared the data with the DYRK1A interactome dataset. Analysis of three independent DCAF7-HA pull-down samples identified 32 proteins specifically detected in all three replicates including DCAF7, DYRK1A and five proteins earlier detected in DYRK1A MudPIT analysis. Given the proposed role of DCAF7 as a scaffold protein, we tested whether DCAF7 mediates its interaction with some of its interacting proteins. Interestingly, we found that RNF169, a recently characterized RING-domain ubiquitin ligase involved in the DNA double-strand break (DSB) repair, binds to N-terminus of DYRK1A independently of DCAF7. Furthermore, our data show that DYRK1A is required for DCAF7-RNF169 interaction because this interaction was abolished in human U-2 OS cells where DYRK1A gene was disrupted using CRISPR-Cas9 editing (DYRK1A-KO cells). Interestingly, stable overexpression of DCAF7 in DYRK1A-KO U-2 OS cells rescued some of the phenotypes observed in these cells, suggesting that DCAF7 could be an effector downstream of DYRK1A. Our findings revealed an unexpected scaffolding role of DYRK1A that is required for mediating the interaction between DNA repair protein RNF169 and the WD-repeat protein DCAF7. While further studies are needed to understand the exact role of the RNF169-DCAF7 interaction in DNA repair, the novel role of DYRK1A as a scaffold protein could explain some aspects of the dosage-dependent function of this protein kinase. In addition, we report here an initial functional characterization of the DCAF7-DYRK1A interaction in human cells and discuss novel functional interactions of DCAF7 that could be independent of DYRK1A. Citation Format: Varsha Anathapadmanabhan, Selene Swanson, Siddharth Saini, Vijay Menon, Larisa Litovchick. Proteomic and functional studies identify DCAF7 as major partner of DYRK1A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2017-343