Yiannis Drosos

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic ‘hits’, compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction.

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

BackgroundIntegrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.ResultsIn the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.ConclusionA model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

NUCKS overexpression in breast cancer.

BackgroundNUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.ResultsThe immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.ConclusionThe results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

Dynamic distribution of claudin proteins in pancreatic epithelia undergoing morphogenesis or neoplastic transformation

Background: The assembly of distinct proteins into tight junctions results in the formation of a continuous barrier that regulates the paracellular flux of water, ions, and small molecules across epithelia. The claudin protein family encompasses numerous major structural components of tight junctions. These proteins specify the permeability characteristics of tight junctions and consequently, some of the physiological properties of epithelia. Furthermore, defective claudin expression has been found to correlate with some diseases, tumor progression, and defective morphogenesis. Investigating the pattern of claudin expression during embryogenesis or in certain pathological conditions is necessary to begin disclosing the role of these proteins in health and disease. Results: This study analyzed the expression of several claudins during mouse pancreas organogenesis and in pancreatic intraepithelial neoplasias of mouse and human origin. Conclusions: Our results underscored a distinctive, dynamic distribution of certain claudins in both the developing pancreas and the pancreatic epithelium undergoing neoplastic transformation. Developmental Dynamics 241:583–594, 2012.

Dynamic expression of the vertebrate-specific protein Nucks during rodent embryonic development.

The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis.

RGD Binding to Integrin Alphavbeta3 Affects Cell Motility and Adhesion in Primary Human Breast Cancer Cultures

Integrins mediate cell adhesion to the extracellular matrix. Integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site and has been associated with high malignant potential in breast cancer cells, signaling the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including RGD peptides, have been used as potential anti-cancer agents. In the present work, the effect of the linear RGD hexapeptide GRGDSP was studied, for the first time, on breast tumor explants, as well as on well-spread human breast cancer cells from primary cultures, using the explant technique, to clarify the role of this peptide in the suppression of breast cancer cell migration. The results showed that incubation of breast tumor explants with RGD peptide at the beginning of culture development inhibited completely the migration of cancer cells out of the tissue fragment as revealed by electron microscopy. RGD incubation of well-spread breast cancer cells from primary culture resulted in rounding and shrinkage of the cells accompanied by altered distribution of integrin alphavbeta3 and concomitant F-actin cytoskeletal disorganization, as revealed by immunofluorescence. Electron immunocytochemistry showed aggregation of integrin alphavbeta3 at the cell periphery and its detection in noncoated vesicles. However, Western immunoblotting showed no change in beta3 subunit expression, despite the altered distribution of the integrin alphavbeta3. In light of the above, it appears that the RGD peptide plays an important role in the modulation of cell motility and in the perturbation of cell attachment affecting the malignant potential of breast cancer cells in primary cultures.

Lack of Prox1 Downregulation Disrupts the Expansion and Maturation of Postnatal Murine β-Cells

Transcription factor expression fluctuates during β-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature β-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with β-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature β-cells has no functional consequences; in contrast, Prox1 overexpression in immature β-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal β-cells. Also, we uncovered substantial deficiency in β-cells that overexpress Prox1 of the key regulator of β-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-βH1 β-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal β-cells.

ATM-deficiency increases genomic instability and metastatic potential in a mouse model of pancreatic cancer

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.

Prox1-Heterozygosis Sensitizes the Pancreas to Oncogenic Kras-Induced Neoplastic Transformation

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.