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Syndecan-1 (SDC1, CD138) is one of the heparan sulfate proteoglycans and is essential for maintaining normal cell morphology, interacting with the extracellular and intracellular protein repertoire, as well as mediating signaling transduction upon environmental stimuli. The critical role of SDC1 in promoting tumorigenesis and metastasis has been increasingly recognized in various cancer types, implying a promising potential of utilizing SDC1 as a novel target for cancer therapy. This review summarizes the current knowledge on SDC1 structure and functions, including its role in tumor biology. We also discuss the highlights and limitations of current SDC1-targeted therapies as well as the obstacles in developing new therapeutic methods, offering our perspective on the future directions to target SDC1 for cancer treatment.

Overlapped genes between Genes upregulated in Ptenpc-/-Smad4pc-/- tumors as compared to Ptenpc-/- tumors ({greater than or equal to}2 fold) and genes upregulated in GFP+ tumors cells from Ptenpc-/-Smad4pc-/- mice as compared to Tomato+ cells ({greater than or equal to}4 fold).

Supplementary Figure S1. CyTOF analysis of biological samples from Ptenpc-/-Smad4pc-/- mice (Related to Figure 2). Supplementary Figure S2. Strategy used for MDSCs Isolation (Related to Figure 3). Supplementary Figure S3. Treatment scheme for Gr-1 antibody, peptibody, and Cxcr2 inhibitor SB225002. Supplementary Figure S4. IHC staining of Ki67, CD45, Vimentin, Smooth muscle actin (SMA) and Trichrome staining of mouse prostate tissues treated with IgG control or Gr1 antibody. Supplementary Figure S5. The top 10 differentially expressed genes in Ptenpc-/-Smad4pc-/- tumors as compared to Ptenpc-/- tumors, identified by microarray analysis (n=5). Figure S6. Top 10 activated oncogenic signatures identified by GSEA analysis in Ptenpc-/- Smad4pc-/- tumors as compared to Ptenpc-/- tumors (n=5). Figure S7. Clustering of primary prostate tumors from Wallace et al into MDSC-high and MDSC-low subtypes.

Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and to identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D-driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low-dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D-driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal endoplasmic reticulum stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC.Significance:This study demonstrates the critical role of glucose metabolism in resistance to MAPK inhibition in KRAS-driven pancreatic cancer, uncovering a potential therapeutic approach for treating this aggressive disease.

Supplementary Tables 1-7.

Supplementary Figures S1-S6.

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K–AKT–MTOR pathway and by the activation of AXL, PDGFRa, and HER1–2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. Cancer Res; 75(6); 1091–101. ©2015 AACR.

supplementary Figures S1-S10. S1 growth and regression of iKRAS tumors. S2 culture comparison. S3 immunohistochemistry of p-erk and p-s6 S4 Phospho rtk array S5 Validation of anti AXL antibodies S6 validationn of the AXL inhibitor in vitro S7 validationn of the AXL inhibitor in vivo S8 efficacy of targeting AXL S9 RTK array of iKRAS cells treated with multiple RTK inhibitors S10 P-ERK levels

supplementary material and method

Activating mutations in KRAS (KRAS*) are present in nearly all pancreatic ductal adenocarcinoma (PDAC) cases and critical for tumor maintenance. By using an inducible KRAS* PDAC mouse model, we identified a deubiquitinase USP21-driven resistance mechanism to anti-KRAS* therapy. USP21 promotes KRAS*-independent tumor growth via its regulation of MARK3-induced macropinocytosis, which serves to maintain intracellular amino acid levels for anabolic growth. The USP21-mediated KRAS* bypass, coupled with the frequent amplification of USP21 in human PDAC tumors, encourages the assessment of USP21 as a novel drug target as well as a potential parameter that may affect responsiveness to emergent anti-KRAS* therapy.

Among the many sarcoma subtypes, high-grade tumors that have failed to differentiate or undergone dedifferentiation strongly correlate with metastatic potential and poor clinical outcomes. To examine the factors that regulate cell fate, lineage plasticity, and tumor grade, we focused on two common liposarcoma variants, dedifferentiated liposarcoma (DDLS) and well-differentiated liposarcomas (WDLS), that exist at both ends of the differentiation spectrum. Since tumor grading using histology is highly subjective due to widespread tumor heterogeneity, we hypothesized that an sc/snRNA-seq-based approach would be more accurate. To determine if candidate therapies modulate differentiation, we developed a quantitative metric of differentiation using scRNA-seq. The resulting mesenchymal tissue landscape (MTL) is akin to a Waddington landscape but optimized to distinguish DDLS from WDLS. After prospectively obtaining tumors from seven chemo-naïve patients, we performed scRNA-seq on three DDLS and five WDLS specimens, totaling 59,917 cells. Classical markers were used to identify cell types. Since no pre-existing liposarcoma training sets existed, malignant cells were identified by copy number variation (CNV) inferred from the scRNA-seq data. Overexpression of MDM2, CDK4, and HMGA2, known markers of LS, were positive in clusters classified by SingleR as fibroblasts, adipocytes, and myocyte - the mesenchymal subpopulation. We also inferred CNVs and found expected amplifications in 12q13-15 in the mesenchymal subpopulation. Next, we measured cell differentiation by CytoTRACE. A subcluster of malignant fibroblast-like cells positive for MDM2 and CDK4 was the most de-differentiated. Conversely, cells labeled as adipocytes and endothelial cells were the most differentiated cells. Mapping WDLS and DDLS onto the MTL demonstrated that WDLS had more mature cells than DDLS. Surprisingly, while the pathology-assigned diagnosis falls into just two categories, our approach identified a wide range of liposarcoma cells spanning the full spectrum of possible differentiation states. Though follow-on studies are needed, our data suggest that quantitative differentiation metrics can identify malignant cells in transit from a DDLS-to-WDLS state, or possibly the reverse, whereby cells undergo dedifferentiation. Both phenomena are suspected clinically, given the frequent co-existence of WDLS and DDLS in the same tumors, and the noted emergence of DDLS from WDLS diagnosed years earlier. Summarizing our key findings, we measured the degree of differentiation in liposarcoma cells as a precursor to ongoing studies aimed at understanding the molecular mechanisms that underpin LS plasticity and differentiation. Ultimately, we aim to identify the genes and pathways linked to cell fate and discover novel biologically targeted therapies capable of pushing DDLS toward a less aggressive, slower-growing state. Citation Format: Danh Truong, Hannah C. Beird, Chia-Chin Wu, Sandhya Krishnan, Davis Ingram, Alexander Lazar, Emily Keung, Christina Roland, Wantong Yao, Robert Benjamin, Neeta Somaiah, Barry W. Feig, Joseph A. Ludwig. Defining differentiation States in well-differentiated and de-differentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6558.

Introduction: DNA replication is a tightly regulated, multi-step process involving licensing and replication factors that faithfully orchestrate a single duplication of the entire genome exclusively during S phase of the cell cycle. Although the mechanisms that prevent uncoordinated DNA replication are well characterized, it remains unclear how cells counteract its potentially harmful effects when it does occur, such as in cancer cells driven by aberrant oncogenic signaling and loss of cell cycle checkpoints. Here, we identify DPY30, an understudied component of all COMPASS complexes, as a modulator of DNA replication. We found upregulated DPY30 expression in pancreatic ductal adenocarcinoma (PDAC) as well as a correlation between increasing DPY30 expression level and higher tumor grade and poor clinical outcomes. Experimental procedure: Human- and mouse-derived PDAC cells silenced or knockout for DPY30 expression were generated and characterized for their in vivo growth in recipient mice with increasing level of immune pressure. Chromatin immunoprecipitation followed by massive parallel sequencing was used to measure the epigenetic landscape in DPY30 or WDR5 knockdown cells. Unbiased proteomics and transcriptomics analyses were performed to elucidate molecular mechanisms. Results: The canonical function of COMPASS complexes is to regulate histone methylation. Unexpectedly, DPY30 loss did not affect histone methylation patterns compared to WDR5 loss, the core member of the COMPASS-like complex. This prompted unbiased proteomics analysis, which showed that DPY30 interacts with members of the DNA helicase, possibly stabilizing the DNA replication fork progression. We found that DPY30 overexpression, by preventing Geminin ubiquitylation, strongly stabilizes its expression, which in turn suppresses Cdt1 activity in late S-phase, buffering the risk of uncoordinated DNA replication in G2/M phase. Consistently, DPY30 loss resulted in DNA re-replication and chromosomal instability, as manifested by DNA fiber and metaphasic spread. In vivo, whole-exome sequencing analysis of DPY30-knockout tumors revealed that DPY30 loss markedly increased mutational burden in immunodeficient mice. In syngeneic models, DPY30 loss strongly inhibited tumor growth, prolonged survival, and increased T cell infiltration of tumors. Moreover, the immune system selectively cleared tumor cells with complex karyotypes, and we observed improved anti-tumor efficacy with DPY30 knockdown and anti-PD-1 treatment compared to either condition alone. Conclusions: In summary, we have identified a previously undescribed function for DPY30 to stabilize the replisome machinery and prevent excessive DNA replication. Our findings indicate that, in PDAC, DPY30 promotes genome stability, thus providing a rationale for targeting DPY30 or its effector proteins in combination with immune-checkpoint inhibitors. Citation Format: Francesca Citron, Luigi Perelli, Yanshuo Chu, Sanjana Srinivasan, I-Lin Ho, Er-Yen Yen, Rutvi Shah, Sergio Attanasio, Luis Castillo Montanez, Mauro Di Pilato, Shan Jiang, Wantong Yao, Sisi Gao, Angela Deem, Giannicola Genovese, Linghua Wang, Virginia Giuliani, Andrea Viale, Giulio F. Draetta. DPY30 loss leads to DNA re-replication and immunoediting in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 303.

Defining tumor cell immune evasion Mouse models used to study cancer often lack a full immune system, allowing implantation of human tumors into the mice. By contrast, naturally evolving tumors must contend with a fully functional immune system and its destruction of some of the cells (see the Perspective by Ho and Wood). Two groups now report studies on mouse models with a fully intact immune system. Martin et al . started with preexisting murine tumor cell lines and examined their continued evolution in vivo, whereas Del Poggetto et al . examined the development of new pancreatic tumors in the context of inflammation, as is often seen in human patients. In each study, the authors found that the immune system exerted a selective pressure on cells that would give rise to tumors, promoting the survival of those that had lost expression of tumor suppressor genes or activated a specific oncogene. The findings suggest a major role for the immune system in driving tumor evolution across multiple types of cancer. —YN

Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries., AsCpf1 is an alternative nuclease to Cas9 for CRISPR mediated genome engineering. Here the authors demonstrate functional genomic screens with AsCpf1 that minimize library size with no loss in gene targeting efficiency.

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2–4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and—in the case of PDAC—the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface—where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth—is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities. In an inducible mouse model of pancreatic ductal adenocarcinoma, the signalling defect that underlies 90% of these tumours causes increased cell-surface expression of syndecan 1, leading to misregulation of macropinocytosis, and linking the defective signalling with nutrient-salvage pathways.

The association between tumors and inflammation is a long-established clinical observation. Although many studies demonstrated that the inflammatory microenvironment can promote tumor growth through the activation of survival and proliferation programs in cancer cells, the reason why inflammation, an evolutionarily conserved response to damage aimed at reestablishing tissue integrity upon injury, might be integral to tumorigenesis still remains unknown. Pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by poor prognosis, represents a distinctive example of cooperation between inflammation and activated oncogenes. Frequently developed in a context of chronic pancreatitis, PDAC is always associated with an inflammatory microenvironment. As supported by a substantial body of evidence across a multitude of experimental models, when occurring in the context of pancreatitis, mutations of KRAS, the universal oncogenic driver of pancreatic cancer, lead to accelerated tumor development inducing the appearance of neoplastic precursor lesions, such as acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which can evolve into invasive tumors. Interestingly, preneoplastic pancreatic alterations, specifically ADM, have been previously identified in acute and chronic pancreatitis in the absence of oncogene activation. Investigating the effects of inflammation on normal pancreatic epithelial cells, we discovered that long after complete resolution a transient inflammatory event primes cells to cooperate with oncogenic KRAS. Indeed, upon recovery from a single acute inflammation, epithelial cells display an enduring adaptive response associated with sustained epigenetic and transcriptional reprogramming. Such adaptation facilitates the prompt reactivation of acinar-to-ductal metaplasia upon subsequent inflammatory events, representing a physiological mechanism for limiting tissue damage via rapid decrease of zymogen production. Because metaplastic lesions are mediated by the activation of MAPK signaling, we demonstrated that activating mutations of KRAS, maintaining an irreversible ADM, are protective against tissue damage in a contest of pancreatitis. Uncovering a new physiologic role of somatic oncogenic mutations in preserving tissue integrity during repeated damages, we propose that KRAS mutations represent a nearly universal event in pancreatic cancer because beneficial and under strong positive selection in the context of recurrent pancreatitis. Citation Format: I-Lin Ho, Edoardo Del Poggetto, Chiara Balestrieri, Er-Yen Yen, Francesca Citron, Rutvi Shah, Shaojun Zhang, Shan Jiang, Wantong Yao, Haoqiang Ying, Giannicola Genovese, Timothy Heffernan, Anirban Maitra, Linghua Wang, Timothy Wang, Giulio Draetta, Alessandro Carugo, Gioacchino Natoli, Andrea Viale. Epithelial memory of resolved inflammation cooperates with oncogenic KRAS to limit tissue damage while promoting pancreatic cancer [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 1327.

Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and to identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D-driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low-dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D-driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal endoplasmic reticulum stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC. Significance: This study demonstrates the critical role of glucose metabolism in resistance to MAPK inhibition in KRAS-driven pancreatic cancer, uncovering a potential therapeutic approach for treating this aggressive disease.

Cancers are highly complex ecosystems composed of molecularly distinct sub-populations of tumor cells, each exhibiting a unique spectrum of genetic features and phenotypes, and embedded within a complex organ context. To substantially improve clinical outcomes, there is a need to comprehensively define inter- and intra-tumor phenotypic diversity, as well as to understand the genetic dependencies that underlie discrete molecular subpopulations. To this end, we integrated CRISPR-based co-dependency annotations with a tissue-specific co-expression network developed from patient-derived models to establish CoDEX, a framework to quantitatively associate gene-cluster patterns with genetic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Using CoDEX, we defined multiple prominent anticorrelated gene-cluster signatures and specific pathway dependencies, both across genetically distinct PDAC models and intratumorally at the single-cell level. Of these, one differential signature recapitulated the characteristics of classical and basal-like PDAC molecular subtypes on a continuous scale. Anchoring genetic dependencies identified through functional genomics within the gene-cluster signature defined fundamental vulnerabilities associated with transcriptomic signatures of PDAC subtypes. Subtype-associated dependencies were validated by feature-barcoded CRISPR knockout of prioritized basal-like-associated genetic vulnerabilities (SMAD4, ILK, and ZEB1) followed by scRNAseq in multiple PDAC models. Silencing of these genes resulted in a significant and directional clonal shift toward the classical-like signature of more indolent tumors. These results validate CoDEX as a novel, quantitative approach to identify specific genetic dependencies within defined molecular contexts that may guide clinical positioning of targeted therapeutics.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable disease characterized by poor survival, dense desmoplastic stroma and activating mutations in KRAS ( Citation Format: Sanjana Srinivasan, Johnathon Rose, Wantong Yao, Sahil Seth, Michael Peoples, Annette Machado, Chieh-Yuan Li, I Lin Ho, Jaewon J. Lee, Paola A. Guerrera, Eiru Kim, Mustafa Syed, Joseph Daniele, Angela K. Deem, Michael Kim, Christopher A. Bristow, Eugene Koay, Giannicola Genovese, Andrea Viale, Timothy P. Heffernan, Anirban Maitra, Traver Hart, Alessandro Carugo, Giulio Draetta. Diversity across the pancreatic ductal adenocarcinoma disease spectrum revealed by network-anchored functional genomics [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 2136.

Pancreatic cancer (PDAC) is one of the deadliest types of human cancers, owing to late stage at presentation and pervasive therapeutic resistance. The extensive tumour heterogeneity, as well as substantial crosstalk between the neoplastic epithelium and components within the microenvironment are the defining features of PDAC biology that dictate the dismal natural history. Recent advances in genomic and molecular profiling have informed on the genetic makeup and evolutionary patterns of tumour progression, leading to treatment breakthroughs in minor subsets of patients with specific tumour mutational profiles. The nature and function of tumour heterogeneity, including stromal heterogeneity, in PDAC development and therapeutic resistance, are increasingly being elucidated. Deep insight has been gained regarding the metabolic and immunological deregulation, which further sheds light on the complex biology and the observed treatment recalcitrance. Here we will summarize these recent achievements and offer our perspective on the path forward. Keywords: Pancreatic cancer, Genetics, Ttumour heterogeneity, Cancer metabolism, Immune, Evasion, Immunotherapy

Objective: Yap1 oncogene is essential for KRAS-induced pancreatic ductal adenocarcinoma (PDAC) initiation. We recently demonstrated that YAP1 is capable of bypassing KRAS-dependency. However, genetic alterations of YAP1 pathway are rare in human PDAC and its role in tumour maintenance remains unclear. This study investigates YAP1 function in transcriptionally distinct subsets of PDAC and explores the molecular mechanisms for YAP1 activation. Design: Conditional Yap1 allele was crossed into LSL-KrasG12D/+; Trp53R172H/+; Mist1-CreERT2+ mice to study the requirement of Yap1 for PDAC development in adult mice. YAP1 function in advanced PDAC was analyzed in human tissues, PDAC cell lines, patient-derived xenograft samples and mouse PDAC cells, using in vitro and in vivo assays and gene expression analyses. WNT5A expression and its effect on YAP1 activation as well as tumorigenic activity was studied in aforementioned human and mouse systems using genetic and pharmacological approaches. The bypass of KRAS-dependency by WNT5A was evaluated in KrasG12D-driven mouse PDAC cells. Results: Yap1 deletion blocked KrasG12D/Trp53R172H/+ induced PDAC development in mice. YAP1 activation correlated with squamous subtype of human PDAC and poor prognosis. YAP1 activation enhanced malignant phenotype and YAP1 depletion suppressed the tumorigenic activity specifically in squamous subtype tumours. WNT5A was significantly overexpressed in squamous subtype tumours and positively correlated with YAP1 activity. Conversely, WNT5A depletion resulted in YAP1 inactivation and inhibition of tumorigenic activity. WNT5A expression enabled cell survival and tumour growth in the absence of oncogenic Kras. Conclusions: YAP1 oncogene is a major driver for squamous subtype PDAC whose activation is mediated by WNT5A overexpression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers, with a five-year survival rate of around 5% to 8%. To date, very few available drugs have been successfully used to treat PDAC due to the poor understanding of the tumor-specific features. One of the hallmarks of pancreatic cancer cells is the deregulated cellular energetics characterized by the “Warburg effect”. It has been known for decades that cancer cells have a dramatically increased glycolytic flux even in the presence of oxygen and normal mitochondrial function. Glycolytic flux is the central carbon metabolism process in all cells, which not only produces adenosine triphosphate (ATP) but also provides biomass for anabolic processes that support cell proliferation. Expression levels of glucose transporters and rate-limiting enzymes regulate the rate of glycolytic flux. Intermediates that branch out from glycolysis are responsible for redox homeostasis, glycosylation, and biosynthesis. Beyond enhanced glycolytic flux, pancreatic cancer cells activate nutrient salvage pathways, which includes autophagy and micropinocytosis, from which the generated sugars, amino acids, and fatty acids are used to buffer the stresses induced by nutrient deprivation. Further, PDAC is characterized by extensive metabolic crosstalk between tumor cells and cells in the tumor microenvironment (TME). In this review, we will give an overview on recent progresses made in understanding glucose metabolism-related deregulations in PDAC.

Drug resistance and relapse remain key challenges in pancreatic cancer. Here, we have used RNA-seq, ChIP-seq, and genome-wide CRISPR analysis to map molecular dependencies of pancreatic cancer stem cells, highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular, the nuclear hormone receptor RORγ, known to drive inflammation and T-cell differentiation, was upregulated during pancreatic cancer progression, and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth, and a marked improvement in survival. Further, a large-scale retrospective analysis in patients revealed that RORγ expression may predict pancreatic cancer aggressiveness, as it positively correlated with advanced disease and metastasis. Collectively, these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells, suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients.

Mitochondrial dysfunction is behind several neurodegenerative diseases, including Alzheimer disease (AD). Accumulation of damaged mitochondria is already observed at the early stages of AD and has been linked to impaired mitophagy, but the mechanisms underlying this alteration are still not fully known. In our recent study, we show that intracellular cholesterol enrichment can downregulate amyloid beta (Aβ)-induced mitophagy. Mitochondrial glutathione depletion resulting from high cholesterol levels promotes PINK1 (PTEN induced kinase 1)-mediated mitophagosome formation; however, mitophagy flux is ultimately disrupted, most likely due to fusion deficiency of endosomes-lysosomes caused by cholesterol. Meanwhile, in APP-PSEN1-SREBF2 mice, an AD mouse model that overexpresses the cholesterol-related transcription factor SREBF2, cholesterol accumulation prompts an oxidative- and age-dependent cytosolic aggregation of the mitophagy adaptor OPTN (optineurin), which prevents mitophagosome formation despite enhanced PINK1-PRKN/parkin signaling. Hippocampal neurons from postmortem brain of AD individuals reproduce the progressive accumulation of OPTN in aggresome-like structures accompanied by high levels of mitochondrial cholesterol in advanced stages of the disease. Overall, these data provide new insights into the impairment of the PINK1-PRKN mitophagy pathway in AD and suggest the combination of mitophagy inducers with strategies focused on restoring the cholesterol homeostasis and mitochondrial redox balance as a potential disease-modifying therapy for AD.

The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo–YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP–TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5–CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. Significance: We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo–YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5–CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival. Cancer Discov; 6(1); 80–95. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 1

Many common instigators drive the processes of inflammation and carcinogenesis. We have found such a common node in Yap1. In the context of cancer, YAP-TEAD complex is important in modulating the immune microenvironment, such that Yap 1-mediated transcriptional upregulation of CXCL5 by the prostate cancer cells in a PTEN/SMAD4 deficient mouse model leads to the recruitment of MDSCs through the interaction with CXCR2 on them. Depletion of MDSCs or pharmacologic inhibition of CXCR2 leads to impediment of cancer progression. In another well-established pancreatic cancer model (PDAC), Yap1/Tead2 governs escape from mutant Kras (G12D) inhibition through cooperative upregulation of E2F transcription factors to activate a cell cycle and DNA replication program. In the context of inflammation, specifically inflammatory bowel disease, telomere dysfunction is shown to activate pAtm/c-Abl-mediated phosphorylation and stabilization of Yap1 upregulating pro-IL-18, a major proinflammatory factor in IBD. This signaling axis cooperates with the gut microbiome stimulating cytosolic receptors causing activation of caspase-1 cleaving pro-IL-18 into mature IL-18. Epithelial IL-18 leads to recruitment of IFNγ-secreting T cells and other immunocytes provoking classical IBD pathology. Consistent with a role for DNA damage signaling driving IBD, newly diagnosed IBD patient samples exhibited elevated expression of pγH2AX, YAP1, Caspase-1, and IL-18 and significantly reduced telomere lengths. Telomerase reactivation in intestinal epithelium or pharmacologic inhibition of Atm, Yap1, or caspase-1 as well as antibiotic treatment of mice dramatically reduced IL-18 and inflammation. Thus, telomere dysfunction-induced activation of the Atm-Yap1-pro-IL-18 pathway identifies DNA damage signaling as a key instigator and promoter of IBD, illuminating novel therapeutic strategies. Citation Format: Guocan Wang, Xin Lu, Deepavali Chakravarti, Avnish Kapoor, Wantong Yao, Haoqiang Ying, Prasenjit Dey, Chiachin Wu, Denise Spring, Pingping Hou, Pingna Deng, Di Zhao, Baoli Hu, Mao Xizeng, Christopher Logothesis, Michael McArthur, Lynda Chin, Alan Wang, Sujun Hua, Hongai Xia, Gerald C Chu, Carol Lim, Eduardo Vilar, Sarah Blutt, Ronald A. DePinho. YAP in cancer and inflammation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA24.

RNA interference and CRISPR/Cas9-based pooled library screens have revolutionized the field of functional genomics. However, currently available pooled library screens face a trade-off between library effectiveness and library complexity. We developed a multiplexed, high-throughput screening strategy based on an optimized AsCpf1 nuclease that minimizes library size without sacrificing gene targeting efficiency. Our AsCpf1-based multiplexed library performed similarly well compared to currently available CRISPR/Cas9 libraries, but with a single polycistronic crRNA clone targeting each gene. With this strategy, we constructed the smallest whole-genome knock-out library available, “Mini-human” for the human genome, which is one-fourth the size of the smallest CRISPR library currently available.

Increasing researches verify that mitochondrial oxidase phosphorylation (OXPHOS) plays remarkable roles in cancer initiation, development, and drug resistance. Our previous study identified that preclinic OXPHOS inhibitor IACS-010759 significantly suppressed cancer cell growth in brain cancer and acute myeloid leukemia (AML) in vivo and in vitro with very low compound concentration. In pancreatic ductal adenocarcinoma (PDAC), there were actually different responses to OXPHOS inhibition in glucose-independent conditions. Using patient-derived xenograft (PDX) model, we identified sensitive and resistant groups to IACS-010759, and there were dramatic metabolic changes to feed-back compound resistance. By metabolomics analysis, we found a novel lipids-signaling activation mechanism that is responsible for the adaptive metabolic resistance. Furthermore, combination of OXPHOS inhibitor and specific lipids pathway inhibitor showed significant cancer cell growth suppression in resistant PDAC types. Our findings uncovered a novel adaptive metabolism in drug resistance and provide new thoughts on therapy strategy in PDAC. Citation Format: Ziheng Chen, Jintan Liu, I-Lin Ho, Johnathan Rose, Liang Yan, Wantong Yao, Andrea Viale, Alessandro Carugo, Haoqiang Ying, Giulio Draetta. Lipids signaling contributes to glucose-independent metabolic adaptation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C12.

Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

Adaptor proteins are involved in the assembly of various intracellular complexes and the regulation of cellular functions. Membrane-associated guanylate kinase inverted 2 (MAGI2), also known as synaptic scaffolding molecule (S-SCAM), plays a critical role in signal transduction by assembling and anchoring its ligands. However, the role of MAGI2 in mediating apoptosis remains largely unknown. In the present study, BEL-7404 human hepatocellular carcinoma cells were transfected with a plasmid containing myc-MAGI2 or an empty plasmid and cell viability was then determined using the Cell Counting kit-8. Apoptosis was also detected using an Annexin V apoptosis assay. The cells were then treated with various doses of staurosporine (STS) for different periods of time. The overexpression of myc-MAGI2 was found to sensitize the BEL-7404 cells to apoptosis in response to STS in a time- and dose-dependent manner. Our results demonstrated that MAGI2 enhanced STS-induced apoptosis by increasing the protein expression of cytoplasmic phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and decreasing its protein degradation. The apoptotic sensitivity of the cells caused by the overexpression of myc-MAGI2 was reversed by the silencing of PTEN expression by PTEN siRNA, thus revealing a momentous role of PTEN in the enhancement of the sensitivity of cancer cells to STS-induced apoptosis by MAGI2. Finally, we observed that the MAGI-PTEN complex triggered by MAGI2 overexpression reduced the phosphorylation levels of AKT. These results suggest that MAGI2 overexpression enhances the sensitivity of cancer cells harboring ectopic PTEN to STS-induced apoptosis.

Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5β1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5β1. These results suggest that the insufficient fibronectin caused by the integrin α5β1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin β1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5β1, and suggested a new target for breast cancer therapy.

Integrin αvβ3 receptor is expressed on several types of cancer cells, including pancreatic cancer cells, and plays an important role in tumor growth and metastasis. The ability to target the integrin αvβ3 receptor on cancer cells increases the efficacy of targeted therapy and reduces the side effects. The aim of this study is to develop a novel arginine-glycine-aspartic acid (RGD) peptide -conjugated albumin nanoparticle to enhance the intracellular uptake of anticancer drug into the pancreatic cancer cells through receptor-mediated endocytosis. In the cellular uptake studies, the fluorescent signal of RGD-conjugated BSANPs in BxPC3 cells was higher than that of BSANPs without RGD conjugation as determined by fluorescence spectrophotometer. We also found that BSANPs bound to BxPC3 cells in a time- and concentration-dependent manner. The uptake of RGD-conjugated BSANPs by pancreatic cancer cells was inhibited by an excess amount of free RGD peptide, indicating that the binding and/or uptake were mediated by the αvβ3 receptor. Furthermore, the nanoparticles were found to be located close to the nuclei by using laser scanning confocal microscopy. Besides, no significant in vitro cytotoxicity was observed as measured with MTT assay. Both in vitro and in vivo antitumor efficacy was improved by targeting gemcitabine-loaded nanoparticles to BxPC-3 cells using RGD peptides. Therefore, the RGD-conjugated BSANPs hold great potential as an effective drug delivery system to deliver therapeutic agents to pancreatic cancer.

SummaryCurrent treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1–4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.

Pancreatic Ductal Adenocarcinoma (PDAC) is an incurable disease characterized by poor survival, a dense desmoplastic stroma, and frequent activating mutations in KRAS ( Citation Format: Johnathon L. Rose, Alessandro Carugo, Wantong Yao, Sahil Seth, Sanjana Srinivasan, Michael Peoples, Traver Hart, Timothy Heffernan, Giulio Draetta. Reverse functional genomics: Identifying differentially regulated functional networks as predictive biomarkers of pivotal vulnerabilities in pancreatic cancer [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 392.

Expression level of integrin alpha5 in tumor cells has been indicated to be involved in cell proliferation and organ-specific metastasis. We previously demonstrated that ITGA5 expression was downregulated in the high invasive MDA-MB-468 cells compared with other breast cancer cell lines. In this study, we found that the methylation status in the region around transcriptional start site of ITGA5 gene was increased in MDA-MB-468 cells. Overexpression of integrin alpha5 on MDA-MB-468 cells resulted in cell growth inhibition, which could be reversed by adhesion to fibronectin. Cell adhesion and spreading to fibronectin was enhanced after ITGA5 was overexpressed in MDA-MB-468 cells, while cell migration was attenuated. Knockdown of ITGA5 in MCF-7 cells led to cell growth inhibition but had little influence on cell migration. These findings indicated the diverse roles of ITGA5 expression in breast cancer cells.

DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

PTEN is a critical tumor suppressor gene mutated frequently in various human cancers. Previous studies have showed that PTEN mRNA expression is down-regulated by TGF-beta1 in various cell lines. In present study, we have found that TGF-beta1 down-regulates PTEN mRNA and protein expression in a dose- and time-dependent manner in hepatocarcinoma cell line SMMC-7721. Based on the PTEN promoter dual-luciferase report assay, we have found that PTEN transcription is not affected by TGF-beta1. By using transcriptional inhibitor actinomycin D (Act D), the turnover rate of PTEN transcripts appeared to be accelerated during TGF-beta1 stimulation, which indicated that down-regulation of PTEN by TGF-beta1 was post-transcriptional. What interested us was that transfection of PTEN coding sequence increased TGF-beta1-induced degradation of PTEN mRNA, suggesting that PTEN coding region was account for TGF-beta1-mediated down-regulation of PTEN. In addition, TGF-beta1 down-regulated PTEN expression was blocked by the TbetaIR inhibitor SB431542 and the p38 inhibitor SB203580, suggesting Smad and p38 MAPK signal pathways played crucial roles in PTEN down-regulation via TGF-beta1 stimulation. In this study, we also found TGF-beta1 accelerated down-regulation of PTEN through the ubiquitin-proteasome pathway. Collectively, our data clearly demonstrated that TGF-beta1-mediated down-regulation of PTEN was post-transcriptional and post-translational, depending on its coding sequence, Smad and p38-MAPK signal pathways were involved in this down-regulation.

Podocytes can influence glomerular endothelial cell (GEnC) barrier properties and take part in the development of proteinuria by some molecules. Angiopoietin-like protein 3 (Angptl3), secreted by podocytes, is a member of the angiopoietin-like protein family that has important biological functions in endothelial cells. In our previous studies, we showed that mRNA expression of Angptl3 increased significantly in kidneys of children with minimal change nephrotic syndrome. And the mRNA level of Angptl3 was increased in the glomerulus of adriamycin rats with the development of proteinuria. It was also found that Angptl3 was expressed in the cytoplasm of cultured podocytes. Thus, Angptl3 might influence the biological functions of GEnCs in a paracrine manner. In this study, we found that Angptl3 could increase the permeability of GEnCs and increase the level of protein kinase B phosphorylation in cultured GEnCs in vitro. LY294002, a phosphatidylinositol-3 kinase inhibitor, could prevent the increase of permeability of GEnCs induced by Angptl3. Our results also indicated that the integrin alphaVbeta3 antibody (LM609) could block the Angptl3-induced protein kinase B phosphorylation.

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K–AKT–MTOR pathway and by the activation of AXL, PDGFRa, and HER1–2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. Cancer Res; 75(6); 1091–101. ©2015 AACR.

Background Tumor cells exhibit abnormal actin remodeling profiles, which involve the altered expressions of several important actin-binding proteins. Profilin1 (Pfn1), originally identified as an actin-associated protein, has been linked to several human malignancies. Our recent studies suggested that Pfn1 facilitates apoptosis in pancreatic cancer cells. Here, we investigated the exact role of Profilin1 (Pfn1) in pancreatic adenocarcinoma (PDAC) and the underlying mechanisms. Methods Pfn1 protein expression in PDAC specimens was analyzed by immunohistochemistry using a tissue microarray (TMA) containing PDAC tumor tissue and corresponding normal tissue samples from 72 patients. The effect of Pfn1 expression on cancer proliferation was assessed in cells by up- and down-regulation of Pfn1 in vitro and in vivo. Immunoprecipitation and mass spectrometry were used to identify the Pfn1-associated proteins and potential pathways. Results Pfn1 was downregulated in clinical pancreatic adenocarcinoma specimens compared with the surrounding benign tissues. Univariate survival analysis of the PDAC cohorts showed that low expression of Pfn1 was significantly correlated with shortened patient survival (mean 14.2 months versus 20.9 months, P

Background A number of oncoproteins and tumor suppressors are known to be neddylated, but whether the neddylation pathway is entirely activated in human cancer remains unexplored. Methods NEDD8-activating enzyme (NAE) (E1) and NEDD8-conjugating enzyme (E2) expression and global-protein neddylation were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction analysis. Cell proliferation, clonogenic survival, migration, and motility in vitro, as well as tumor formation and metastasis in vivo, were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Survival was analyzed with Kaplan-Meier methods and compared by the log-rank test. All statistical tests were two-sided. Results The entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global-protein neddylation, is overactivated in both lung adenocarcinoma and squamous-cell carcinoma. Compared with lung adenocarcinoma patients with low expression, those with high expression had worse overall survival (NEDD8-activating enzyme E1 subunit 1 [NAE1]: hazard ratio [HR] = 2.07, 95% confidence interval [CI] = 0.95 to 4.52, P = .07; ubiquitin-conjugating enzyme E2M (UBC12): HR = 13.26, 95% CI = 1.77 to 99.35, P = .01; global protein neddylation: HR = 3.74, 95% CI = 1.65 to 8.47, P = .002). Moreover, inhibition of neddylation by the NAE inhibitor MLN4924 statistically significantly suppressed proliferation, survival, migration, and motility of lung cancer cells in vitro and tumor formation and metastasis in vivo. At the molecular level, MLN4924 inactivated Cullin-RING E3 ligases, led to accumulation of tumor-suppressive Cullin-RING E3 ligase substrates and induced phorbol-12-myristate-13-acetate-induced protein 1 (NOXA)-dependent apoptosis or cellular senescence. Conclusions Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target.

Pancreatic cancer has an extremely poor prognosis mainly due to lack of effective treatment options. Radiotherapy is mostly applied to locally advanced cases, although tumor radioresistance limits the effectiveness. Profilin1, a novel tumor suppressor gene, was reported to be down-regulated in various cancers and associated with tumor progression. The objective of this study was to demonstrate how profilin1 affected pancreatic cancer radiosensitivity. We showed profilin1 was down-regulated in pancreatic cancer cells after exposure to radiation, and re-expression of profilin1 suppressed tumor cell viability and increased DNA damage following irradiation. Further studies revealed that up-regulation of profilin1 facilitated apoptosis and repressed autophagy induced by irradiation, which might sensitize pancreatic cancer cells to radiation treatment. Our findings may provide a novel therapeutic strategy for sensitizing pancreatic cancer to radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.