Nicole M. Aiello

Robust cellular reprogramming occurs spontaneously during liver regeneration

Cellular reprogramming-the ability to interconvert distinct cell types with defined factors-is transforming the field of regenerative medicine. However, this phenomenon has rarely been observed in vivo without exogenous factors. Here, we report that activation of Notch, a signaling pathway that mediates lineage segregation during liver development, is sufficient to reprogram hepatocytes into biliary epithelial cells (BECs). Moreover, using lineage tracing, we show that hepatocytes undergo widespread hepatocyte-to-BEC reprogramming following injuries that provoke a biliary response, a process requiring Notch. These results provide direct evidence that mammalian regeneration prompts extensive and dramatic changes in cellular identity under injury conditions.

Upholding a role for EMT in pancreatic cancer metastasis

Epithelial-to-mesenchymal transition (EMT) is a cellular program that operates in the context of embryogenesis, wound-healing and carcinoma pathogenesis to drive epithelial cells toward a mesenchymal state. During carcinoma progression, EMT enables the cells forming these tumours to acquire the traits of highly malignant cells, notably motility, invasiveness and an ability to disseminate to form distant metastases. Indeed, a number of published reports have associated EMT with a variety of malignant carcinoma cells. Recently, however, Zheng et al. (1) reported that in genetically engineered mouse models of pancreatic adenocarcinoma development, carcinoma cells could metastasize without activating EMT programs. Their conclusions, if sustained by the evidence presented, would prompt a major change in how we conceptualize malignant progression and metastasis of carcinoma cells, including the neoplastic cells in human carcinomas.

Metastatic progression is associated with dynamic changes in the local microenvironment

Most cancer-associated deaths result from metastasis. However, it remains unknown whether the size, microenvironment or other features of a metastatic lesion dictate its behaviour or determine the efficacy of chemotherapy in the adjuvant (micrometastatic) setting. Here we delineate the natural history of metastasis in an autochthonous model of pancreatic ductal adenocarcinoma (PDAC), using lineage tracing to examine the evolution of disseminated cancer cells and their associated microenvironment. With increasing size, lesions shift from mesenchymal to epithelial histology, become hypovascular and accumulate a desmoplastic stroma, ultimately recapitulating the primary tumours from which they arose. Moreover, treatment with gemcitabine and nab-paclitaxel significantly reduces the overall number of metastases by inducing cell death in lesions of all sizes, challenging the paradigm that PDAC stroma imposes a critical barrier to drug delivery. These results illuminate the cellular dynamics of metastatic progression and suggest that adjuvant chemotherapy affords a survival benefit by directly targeting micrometastases.

Doublecortin-like kinase 1 is elevated serologically in pancreatic ductal adenocarcinoma and widely expressed on circulating tumor cells.

Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.

Echoes of the embryo: using the developmental biology toolkit to study cancer

ABSTRACT The hallmark of embryonic development is regulation – the tendency for cells to find their way into organized and ‘well behaved’ structures – whereas cancer is characterized by dysregulation and disorder. At face value, cancer biology and developmental biology would thus seem to have little to do with each other. But if one looks beneath the surface, embryos and cancers share a number of cellular and molecular features. Embryos arise from a single cell and undergo rapid growth involving cell migration and cell-cell interactions: features that are also seen in the context of cancer. Consequently, many of the experimental tools that have been used to study embryogenesis for over a century are well-suited to studying cancer. This article will review the similarities between embryogenesis and cancer progression and discuss how some of the concepts and techniques used to understand embryos are now being adapted to provide insight into tumorigenesis, from the origins of cancer cells to metastasis. Summary: This article reviews the similarities between embryogenesis and cancer progression and discusses how the concepts and techniques of developmental biology are being applied to provide insight into all aspects of tumorigenesis.

Combining Machine Learning and Nanofluidic Technology To Diagnose Pancreatic Cancer Using Exosomes

Circulating exosomes contain a wealth of proteomic and genetic information, presenting an enormous opportunity in cancer diagnostics. While microfluidic approaches have been used to successfully isolate cells from complex samples, scaling these approaches for exosome isolation has been limited by the low throughput and susceptibility to clogging of nanofluidics. Moreover, the analysis of exosomal biomarkers is confounded by substantial heterogeneity between patients and within a tumor itself. To address these challenges, we developed a multichannel nanofluidic system to analyze crude clinical samples. Using this platform, we isolated exosomes from healthy and diseased murine and clinical cohorts, profiled the RNA cargo inside of these exosomes, and applied a machine learning algorithm to generate predictive panels that could identify samples derived from heterogeneous cancer-bearing individuals. Using this approach, we classified cancer and precancer mice from healthy controls, as well as pancreatic cancer patients from healthy controls, in blinded studies.

Orthotopic Injection of Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma is an aggressive disease with a 5-yr survival rate of only 5%. The location of the pancreas in the abdomen, where it is obscured by other organs, makes it a difficult tissue to study and manipulate. This protocol describes in detail how to orthotopically inject cancer cells into the pancreas in mice. This technique is particularly useful when the cells must be manipulated in ways that cannot be modeled genetically.

Abstract B40: Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma

Deregulation of cellular epigenetics is essential for malignant transformation. However, the mechanisms that cause epigenetic alterations in cancer cells are incompletely understood. Recent evidence has shown that cellular metabolism has a direct impact on the epigenome, since many chromatin-modifying enzymes rely on intracellular metabolites as cofactors or donor substrates. We have previously shown that the metabolic enzyme ATP-citrate lyase (ACLY), which generates nuclear-cytoplasmic acetyl-CoA from glucose, is required for maintaining histone acetylation levels in multiple mammalian cell types, suggesting that alterations in acetyl-CoA metabolism in cancer cells might also impact histone acetylation levels. Cellular metabolism is massively rewired in multiple cancer types, including pancreatic cancer, although the impact of metabolic alterations on the tumor epigenome is poorly understood. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. In this study, we demonstrate that acetyl-CoA abundance in cancer cells is dynamically regulated by glucose availability and that histone acetylation levels are responsive to the ratio of acetyl-CoA:coenzyme A within the nucleus. To test whether oncogene activation could mediate changes in histone acetylation in vivo, we performed immunohistochemical analysis comparing pancreata from mice expressing Kras G12D with those from mice with WT Kras. Whereas the acinar cells of WT mice exhibited very low levels of histone H4 (K5/8/12/16) acetylation, in KPC (LSL-KrasG12D; p53L/+; Pdx1-Cre) mice, acinar H4 acetylation was markedly increased, prior to the appearance of histological abnormalities or aberrant cell proliferation. High H4 acetylation persisted in pancreatic intraepithelial neoplasia (PanIN) and PDA. Histone acetylation in PanIN-derived primary cells was selectively impaired by PI3K and Akt inhibitors, correlating with suppression of glucose consumption and cellular acetyl-CoA levels. Moreover, addition of supraphysiological doses of acetate, a source of acetyl-CoA alternative to glucose, restored histone acetylation levels. These data suggest that oncogenic Kras promotes elevated histone acetylation preceding tumor development through Akt-dependent regulation of cellular acetyl-CoA levels. Further mechanistic analysis suggests that Akt promotes elevated histone acetylation through combined effects on promoting glucose uptake and phosphorylation and activation of ATP-citrate lyase, a metabolic enzyme that produces nuclear-cytoplasmic acetyl-CoA. The aberrant activation of the PI3K/Akt pathway occurs in broad variety of human malignancies. pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors, suggesting that PI3K-Akt-dependent promotion of acetyl-CoA metabolism may contribute to histone acetylation levels in multiple cancer types. Our data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in tumors and offer novel insights on Kras-induced pancreatic carcinogenesis. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nathaniel W. Snyder, Ellen Jackson, Nicole M. Aiello, Benjamin A. Garcia, Lewis A. Chodosh, Ben Z. Stanger, Ian A. Blair, Kathryn E. Wellen. Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B40.

Abstract PR03: Exploring the link between Kras and histone acetylation

KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) have been shown to extensively rewire cellular metabolism to promote macromolecular biosynthesis and maintain redox homeostasis. Recently, compelling evidence has emerged demonstrating that many epigenetic modifications are sensitive to the availability of cellular metabolites. For example, acetyl-CoA is the donor substrate for lysine acetylation, and histone acetylation is responsive to levels of acetyl-CoA, which is produced largely from glucose metabolism. We thus hypothesized that oncogenic metabolic reprogramming alters metabolite levels in a way that impacts the epigenome and could thus contribute to tumorigenesis. Indeed, we find that oncogenic Akt activation promotes elevated histone acetylation, and that this effect is mediated by ATP-citrate-lyase (ACLY), a nucleocytoplasmic enzyme that converts glucose-derived citrate into acetyl-CoA. In order to understand what cellular processes are affected by such AKT-ACLY-mediated increase in histone acetylation, we employed a mouse model of pancreatic ductal adenocarcinoma, driven by oncogenic Kras (Pdx1-Cre; LSL-KrasG12D; TP53L/+). In this model, the PI3K/Akt pathway is activated downstream of Kras. By immunohistochemistry we found that, in mice with WT Kras, pancreatic acinar cells exhibit very low levels of histone H4 (K5/8/12/16) acetylation (AcH4), although AcH4 is clearly detectable in ductal epithelial cells and islets. By contrast, in mice expressing oncogenic Kras in the pancreas (LSL-KrasG12D; p53L/+; Pdx1-Cre), acinar H4 acetylation is dramatically increased. Remarkably, this overt increase in histone acetylation precedes the appearance of histological abnormalities and persists during several steps in tumor progression. Moreover, Kras and Akt-dependent reuglation of histone acetylation levels can be recapitulated in ex vivo acinar cell culture, suggesting that this is a primary effect of oncogene activation in these cells. Akt inhibition also significantly reduced glucose consumption and phosphorylation of ACLY and ultimately decreased acetyl-CoA levels. Akt inhibition in vitro reduces Kras-induced acinar cell hyperacetylation and block acinar-to-ductal metaplasia, an initiating event of pancreatic carcinoma. Our data indicate that Akt orchestrates a metabolic rewiring in pancreatic tumorigenesis, which promotes histone hyperacetylation, a phenotype evident before the manifestation of the disease and that might conceivably contribute to cell plasticity and tumor progression. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nicole M. Aiello, Nathaniel W. Snyder, Andrew J. Worth, Ian A. Blair, Ben Z. Stanger, Kathryn E. Wellen. Exploring the link between Kras and histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR03.

Abstract 2676: Epithelial-mesenchymal transition does not require transcriptional repression of the epithelial program in vivo

Epithelial-mesenchymal transition (EMT), the process by which an epithelial cell loses its adhesion to neighboring cells and acquires a motile, fibroblast-like phenotype, is critical for many aspects of embryogenesis and has been strongly implicated in tumor cell invasion and metastasis. EMT is thought to be primarily regulated at the transcriptional level through repression of epithelial genes by transcription factors such as Snail, Zeb and Twist. To date, the mechanisms driving EMT have been parsed out almost exclusively in vitro under pre-defined conditions (ie, using TGFβ as an EMT inducer), but it remains to be seen whether the machinery that regulates in vitro EMT is physiologically relevant. Using a lineage labeled mouse model of pancreatic ductal adenocarcinoma (PDAC), we isolated tumor cells that have spontaneously undergone EMT to interrogate the molecular mechanisms driving this process in vivo. By comparing the transcriptomes of epithelial (E-cadherin+) and mesenchymal (E-cadherin-) tumor cells, we found that for a majority of PDAC tumors, EMT does not require transcriptional repression of the epithelial program. These cells were fully capable of upregulating a mesenchymal program, as evidenced by the increased expression of collagens, SPARC, MMP2, PDPN, and PRRX1; however, despite the loss of E-cadherin protein, we detected no change in transcript levels of E-cadherin or other epithelial genes after EMT. Our results suggest that in vivo, cells that undergo EMT retain epithelial gene transcription, which may confer the ability to rapidly oscillate between epithelial and mesenchymal states. Citation Format: Nicole M. Aiello, David Balli, Robert Norgard, Jinyang Li, Amine Sahmoud, Ben Z. Stanger. Epithelial-mesenchymal transition does not require transcriptional repression of the epithelial program in vivo . [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2676.