Organoid Models of Human and Mouse Ductal Pancreatic Cancer

The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-α-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-α/β pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein–protein networks.

https://www.pnas.org/content/pnas/106/25/10171.full.pdf

Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.

/pdf/global-analysis-of-cdk1-substrate-phosphorylation-sites-2jxvv5wzgh.pdf

Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

/pdf/single-cell-rna-seq-highlights-intra-tumoral-heterogeneity-woypbkmyfy.pdf

Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma

In eukaryotic cells, prereplication complexes (pre-RCs) are assembled on chromatin in the G1 phase, rendering origins of DNA replication competent to initiate DNA synthesis. When DNA replication commences in S phase, pre-RCs are disassembled, and multiple initiations from the same origin do not occur because new rounds of pre-RC assembly are inhibited. In most experimental organisms, multiple mechanisms that prevent pre-RC assembly have now been identified, and rereplication within the same cell cycle can be induced through defined perturbations of these mechanisms. This review summarizes the diverse array of inhibitory pathways used by different organisms to prevent pre-RC assembly, and focuses on the challenge of understanding how in any one cell type, various mechanisms cooperate to strictly enforce once per cell cycle regulation of DNA replication.

/pdf/strength-in-numbers-preventing-rereplication-via-multiple-5cwk9eni8s.pdf

Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells

Pancreatic ductal adenocarcinoma (PDA) develops through distinct precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). However, genetic features resulting in IPMN-associated PDA (IPMN-PDA) versus PanIN-associated PDA (PanIN-PDA) are largely unknown. Here we find that loss of Brg1, a core subunit of SWI/SNF chromatin remodelling complexes, cooperates with oncogenic Kras to form cystic neoplastic lesions that resemble human IPMN and progress to PDA. Although Brg1-null IPMN-PDA develops rapidly, it possesses a distinct transcriptional profile compared with PanIN-PDA driven by mutant Kras and hemizygous p53 deletion. IPMN-PDA also is less lethal, mirroring prognostic trends in PDA patients. In addition, Brg1 deletion inhibits Kras-dependent PanIN development from adult acinar cells, but promotes Kras-driven preneoplastic transformation in adult duct cells. Therefore, this study implicates Brg1 as a determinant of context-dependent Kras-driven pancreatic tumorigenesis and suggests that chromatin remodelling may underlie the development of distinct PDA subsets.

/pdf/the-chromatin-regulator-brg1-suppresses-formation-of-hmzih654op.pdf

The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma

Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.

/pdf/cdk-phosphorylation-of-a-novel-nls-nes-module-distributed-2g4rdfx77q.pdf

CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication.

Evolutionary change in gene regulation is a key mechanism underlying the genetic component of organismal diversity. Here, we study evolution of regulation at the posttranslational level by examining the evolution of cyclin-dependent kinase (CDK) consensus phosphorylation sites in the protein subunits of the pre-replicative complex (RC). The pre-RC, an assembly of proteins formed during an early stage of DNA replication, is believed to be regulated by CDKs throughout the animals and fungi. Interestingly, although orthologous pre-RC components often contain clusters of CDK consensus sites, the positions and numbers of sites do not seem conserved. By analyzing protein sequences from both distantly and closely related species, we confirm that consensus sites can turn over rapidly even when the local cluster of sites is preserved, consistent with the notion that precise positioning of phosphorylation events is not required for regulation. We also identify evolutionary changes in the clusters of sites and further examine one replication protein, Mcm3, where a cluster of consensus sites near a nucleocytoplasmic transport signal is confined to a specific lineage. We show that the presence or absence of the cluster of sites in different species is associated with differential regulation of the transport signal. These findings suggest that the CDK regulation of MCM nuclear localization was acquired in the lineage leading to Saccharomyces cerevisiae after the divergence with Candida albicans. Our results begin to explore the dynamics of regulatory evolution at the posttranslational level and show interesting similarities to recent observations of regulatory evolution at the level of transcription.

Regulatory evolution in proteins by turnover and lineage-specific changes of cyclin-dependent kinase consensus sites.

Many research groups are engaged in using human pluripotent stem cells (hPSCs) to generate surrogate pancreatic β-cells for transplantation into diabetic patients. However, to our knowledge, there is no report on the successful generation of glucose-responsive insulin-producing β-cells from hPSCs in vitro. Below, we outline a method that is based on published protocols as well as our own experience by which one can differentiate hPSCs along the pancreatic lineage to generate insulin-producing β-cell-like cells. The protocol, which spans five distinct stages, is an attempt to recapitulate the derivation of pancreatic β-cells in vitro as they form in the developing embryo. We included details on materials and techniques, suggest ways to customize it to your hPSC line of choice, added notes on how to monitor and analyze the cells during differentiation, and indicate what results can be expected.

Muluye E. Liku

Papers

The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma

CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication.

Regulatory evolution in proteins by turnover and lineage-specific changes of cyclin-dependent kinase consensus sites.

Directed differentiation of human pluripotent stem cells along the pancreatic endocrine lineage.