Autophagy buffers Ras-induced genotoxic stress enabling malignant transformation in keratinocytes primed by human papillomavirus

Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRas^G12V activity in E6E7–HPV–keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRas^G12V.Subject terms: Cancer models, Macroautophagy, Stress signalling, Mechanisms of disease     

Unlimited division potential of precancerous mouse mammary cells after spontaneous or carcinogen-induced transformation.

Serial transplantation of normal mouse mammary gland in young, isogenic hosts results in progressive loss of division potential, and the transplant line is eventually lost. This is interpreted as an expression of senescence at the cell and tissue level, and it inevitably occurs even though experimental conditions for growth are judged to be optimal. An indefinite extension of mammary growth span can be accomplished by transformation of these normal cells into precancerous cell types, which grow as a benign tissue but which may, however, occasionally undergo a second transformation into a malignant carcinoma. All precancerous tissues tested displayed unlimited growth potential, regardless of whether they occurred spontaneously, or were induced by oncogenic viruses or by administration of chemical carcinogens. Precancerous tissues of both ductal and lobuloalveolar morphology grew continuously. These results indicate that release from cell aging, as measured by the acquisition of unlimited growth potential, is associated with the precancerous state per se, and occurs as an early event in the transition from normal to malignant mammary cells.     

The DNA Damage Signaling Pathway Connects Oncogenic Stress to Cellular Senescence

The mechanisms of tumor suppression must be linked to the oncogenic threats that may affect a normal cell. An important cancer causing mechanism is the accidental activation of genes that stimulate cell proliferation (oncogenes) by a variety of endogenous or environmental mutagens. This event has been experimentally modelled by enforcing the expression of oncogenes in primary cells. The astonishing outcome of these manipulations is that oncogenes trigger antiproliferative responses preventing progression to malignant transformation. These responses bring to an end proliferation due to cell death or a permanent cell cycle arrest called senescence. Here we review evidence indicating that oncogene induced senescence (OIS) involves activation of p53 via the DNA damage response (DDR). These results imply mechanisms of DNA damage in cells expressing oncogenes, that may be secondary to reactive oxygen species and/or some form of “oncogenic stress” that affect normal DNA replication. Interestingly, DNA damage signals persist in cells that escape from senescence. The implications of these signals for tumorigenesis are also discussed. Given that DNA damage signals have now been observed in cells treated with any stimuli known to induce senescence, the process can be redefined as a metabolically viable but permanent cell cycle arrest with persistent DNA damage signaling.     

Acute pancreatitis markedly accelerates pancreatic cancer progression in mice expressing oncogenic Kras

Chronic pancreatitis increases by 16-fold the risk of developing pancreatic ductal adenocarcinoma (PDAC), one of the deadliest human cancers. It also appears to accelerate cancer progression in genetically engineered mouse models. We now report that in a mouse model where oncogenic Kras is activated in all pancreatic cell types, two brief episodes of acute pancreatitis caused rapid PanIN progression and accelerated pancreatic cancer development. Thus, a brief inflammatory insult to the pancreas, when occurring in the context of oncogenic Kras^G12D, can initiate a cascade of events that dramatically enhances the risk for pancreatic malignant transformation.     

High Endogenous Expression of Chitinase 3-Like 1 and Excessive Epithelial Proliferation with Colonic Tumor Formation in MOLF/EiJ Mice

Colorectal cancer (CRC) development is mediated by uncontrolled survival and proliferation of tumor progenitor cells. Using animal models to identify and study host-derived factors that underlie this process can aid interventions in preventing tumor expansion and metastasis. In healthy steady states in humans and mice (e.g. C57BL/6 strain), colonic Chitinase 3-like 1 (CHI3L1) gene expression is undetectable. However, this expression can be induced during intestinal inflammation and tumorigenesis where CHI3L1 plays an important role in tissue restitution and cell proliferation. Here, we show that a wild-derived mouse strain MOLF/EiJ expresses high levels of colonic epithelial CHI3L1 at the steady state due to several nucleotide polymorphisms in the proximal promoter regions of the CHI3L1 gene. Interestingly, these mice spontaneously developed polypoid nodules in the colon with signs of immune cell infiltrations at steady state. The CHI3L1 positive colonic epithelial cells were highly proliferative and exhibited malignant transformation and expansion when exposed in vivo to azoxymethane, one of the well-known colonic carcinogens.     

The cytoplasmic tail peptide sequence of membrane type-1 matrix metalloproteinase (MT1-MMP) directly binds to gC1qR,a compartment-specific chaperone-like regulatory protein

p204 overexpression in retinoblastoma (Rb)-/- mouse embryo fibroblasts or transfection of p204 mutated at both Rb-binding sites confer growth advantages, resulting in a significantly higher number of foci in a cell focus assay. To investigate the possibility that mutated p204 acquires malignant transformation capability, NIH3T3 cells were stably transfected with the expression vector pRcRSV204 double-mutant (p204dm) harboring both the C-terminal deletion up to amino acid 568 and the point mutation from glutamic acid to lysine at position 427, and analyzed for markers typical of cell immortalization and transformation. We detected a greater abundance of cell colonies in soft agar with p204dm-expressing cells than vector control cells. The p204dm-transfected cells also displayed two other characteristics associated with malignant transformation, i.e. growth under low-serum conditions and formation of tumors in athymic nude mice. Moreover, their telomerase activity was significantly higher than in the vector control cells. It would thus seem that p204, devoid of functional Rb-binding motifs, can become oncogenic.     

The Process of Metastases,Formation by Melanoma Patients during the Aftercare – Modelling with Markov Chains and Cox's Regression

It is shown that the metastases states of patients with malignant melanoma during their (usually annual) postoperative care can be considered as a first order Markov chain if the state space is suitably extended. Thus, sojourn times in a state up to transition into another one can be analysed by means of Cox's proportional hazard model to find significant factors influencing these times.     

Inhibition of autophagy impairs tumor cell invasion in an organotypic model

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.     

The steady state kinetics of some biological systems: III. Thermodynamic aspects

Some thermodynamic aspects of steady systems are considered. The time rates of changes, “flux”, of various thermodynamic quantities are formulated. In particular the free energy flux in the steady state, the difference between the free energy flux in the steady and time dependent states and the change in free energy flux upon transition between steady states are discussed. Equations are derived which exhibit the formal similarities and differences between the free energy flux and the conventional free energy change. The temperature dependence of the steady state rate is examined and conditions for “mastery” by a single step discussed. A brief discussion of the role ofrate in the coupling of exergonic and endergonic reactions is given.     

Protein tyrosine phosphatases in cell transformation

The role of tyrosine phosphorylation in cell transformation has been well established. It has been proposed that protein tyrosine phosphatases (PTPases) may be capable of dephosphorylating critical substrates involved in the transformation process, suggesting that they represent a tumor suppressor family of enzymes. Indeed, recent work showed that overexpression of some PTPases in malignant cells counteracted the action of oncogenic tyrosine kinases although overexpression of other forms of these enzymes increased tumorigenicity. The work described herein has provided some insight into the action, both antagonistic and synergistic, of the kinases and phosphatases on cell growth and transformation.     

Gab1 is required for cell cycle transition, cell proliferation, and transformation induced by an oncogenic met receptor

We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cγ binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met–mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor.     

Cell adhesion-mediated transformation of a human SCLC cell line is associated with the development of a normal phenotype

Small cell lung carcinoma (SCLC) is a highly metastatic disease with a poor prognosis due to its resistance to current modes of therapy. SCLC cells appear to arise by oncogenic transformation of self-renewing pulmonary neuroendocrine cells, which have the potential to differentiate into a variety of lung epithelial cell lineages. Epithelial-mesenchymal conversion involved in such cell type transitions leads to the acquisition of an invasive and metastatic phenotype and may be critical for neoplastic progression and its eventual resistance to therapy. In order to investigate mechanisms involved in such transitions, a SCLC cell line was exposed to 5-bromodeoxyuridine. This treatment induced a dramatic conversion from non-substrate-adherent aggregates to monolayers of cells exhibiting an epithelioid phenotype. The phenotypic transition was concomitant with downregulation of vimentin, upregulation of cytokeratins, and cell-cell and cell-matrix adhesion molecules as well as redistribution of the actin cytoskeleton. The changes in the levels and organization of cell-cell and cell-matrix adhesion molecules were correlated with an in vivo loss of tumorigenicity.     

MKK7 and ARF

Sensing, integrating, and processing of stressogenic signals must be followed by accurate differential response(s) for a cell to survive and avoid malignant transformation. The DNA damage response (DDR) pathway is vital in this process, as it deals with genotoxic/oncogenic insults, having p53 as a nodal effector that performs most of the above tasks. Accumulating data reveal that other pathways are also involved in the same or similar processes, conveying also to p53. Emerging questions are if, how, and when these additional pathways communicate with the DDR axis. Two such stress response pathways, involving the MKK7 stress-activated protein kinase (SAPK) and ARF, have been shown to be interlocked with the ATM/ATR-regulated DDR axis in a highly ordered manner. This creates a new landscape in the DDR orchestrated response to genotoxic/oncogenic insults that is currently discussed.     

Effectiveness factors for substrate and product inhibition

The transformation technique of Na and Na (Math. Biosci., 6 , 25, 1970) is extended to convert boundary-value problems involving the steady-state diffusion equation for spherical immobilized enzyme particles exhibiting substrate and product inhibition to initial-value problems. This allows a study of the influence of external mass transfer resistances on the effectiveness factors. It also considerably reduces the number of calculations required to investigate the effect of changes in the kinetic parameters on the overall rate of reaction. The existence of multiple steady states for substrate inhibition kinetics in spherical catalyst particles is illustrated and a criterion for uniqueness of steady states is developed. Effectiveness factors for competitive and noncompetitive product inhibition increase with increasing value of the Sherwood number for the substrate and increasing value of the ratio of substrate to product effective diffusivities within the particle.     

生长因子及其受体在淋巴瘤中异常表达的研究进展

生长因子是一类与受体结合后可以促进细胞增殖和调节细胞多项功能的多肽分子。生长因子及其受体信号通路包括Ras/MAPK、PI3K/AKT和STAT等不仅调控正常细胞的生物学行为,对恶性肿瘤细胞增殖、分化、转化和迁移也具有重要意义。研究发现多种生长因子如VEGF、PDGF和IGF及其受体在多种实体肿瘤如肺癌、乳腺癌、结肠癌中发现有异常表达,在淋巴瘤如DLBCL、PTCL、ML和NL中也存在异常的共同表达,提示在淋巴瘤中可能构成生长因子及其受体的自分泌/旁分泌环路。生长因子及其受体的表达对淋巴瘤患者的预后有一定指导意义,临床研究发现表达生长因子或其受体阳性患者比表达阴性患者有较差的临床预后。这可能与生长因子及其受体对淋巴瘤细胞的增殖、转移和耐药调控有关。目前生长因子及其受体已成为潜在的药物靶点,多种生长因子及其受体抑制剂在开发和临床试验中。本文就近年来生长因子及其受体在淋巴瘤中异常表达研究进展作简要综述。     

Computational Multiple Steady States of the Penicillin G Acylase‐Catalyzed Hydrolysis in an Isothermal CFSTR

A family of an enzymatically catalyzed reaction network was studied, which involves the hydrolysis of penicillin G by penicillin G acylase in an isothermal continuous flow stirred tank reactor (CFSTR). This system consisted of 10 coupled non‐linear equations and was found to be capable of exhibiting computational multiple steady states. A set of kinetic parameters determined from the existing experimental data were used to compute a set of rate constants and two corresponding steady states. This suggested that multiple steady states may occur in the system studied. The phenomena of bistability, hysteresis and bifurcation were discussed. Moreover, the capacity of steady state multiplicity was extended to its family of reaction networks.     

Inhibition of autophagy impairs tumor cell invasion in an organotypic model

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.