Nataly Kravchenko-Balasha

Status of p53 in human cancer cells does not predict efficacy of CHK1 kinase inhibitors combined with chemotherapeutic agents

DNA damage checkpoints cause cell cycle arrest, allowing DNA repair before resumption of the cell cycle. These checkpoints can be activated through several signaling pathways. Checkpoint activators include p53, checkpoint kinase 1 (CHK1), checkpoint kinase 2 and/or MAPKAP kinase 2 (MK2). Many cancer cells lack p53 activity and, therefore, depend on alternative checkpoint activators to arrest the cell cycle following DNA damage. Inhibition of these pathways is expected to specifically sensitize these p53-deficient cells to DNA damage caused by chemotherapy. Using isogenic p53-proficient and p53-deficient cancer cell lines, we show that inactivation of CHK1, but not MK2, abrogates cell cycle arrest following chemotherapy, specifically in p53-deficient cells. However, we show that CHK1 is required to maintain genome integrity and cell viability, and that p53-proficient cells are no less sensitive than p53-deficient cells to CHK1 inhibition in the presence of DNA damage. Thus, combining CHK1 inhibition with DNA damage does not lead to preferential killing of p53-deficient over p53-proficient cells, and inhibiting CHK1 does not appear to be a promising approach for potentiation of cancer chemotherapy.

Information-theoretic analysis of phenotype changes in early stages of carcinogenesis

Cancer is a multistep process characterized by altered signal transduction, cell growth, and metabolism. To identify such processes in early carcinogenesis we use an information theoretic approach to characterize gene expression quantified as mRNA levels in primary keratinocytes (K) and human papillomavirus 16 (HPV16)-transformed keratinocytes (HF1 cells) from early (E) and late (L) passages and from benzo(a)pyrene-treated (BP) L cells. Our starting point is that biological signaling processes are subjected to the same quantitative laws as inanimate, nonequilibrium chemical systems. Environmental and genomic constraints thereby limit the maximal thermodynamic entropy that the biological system can reach. The procedure uncovers the changes in gene expression patterns in different networks and defines the significance of each altered network in the establishment of a particular phenotype. The development of transformed HF1 cells is shown to be represented by one major transcription pattern that is important at all times. Two minor transcription patterns are also identified, one that contributes at early times and a distinguishably different pattern that contributes at later times. All three transcription patterns defined by our analysis were validated by gene expression values and biochemical means. The major transcription pattern includes reduced transcripts participating in the apoptotic network and enhanced transcripts participating in cell cycle, glycolysis, and oxidative phosphorylation. The two minor patterns identify genes that are mainly involved in lipid or carbohydrate metabolism.

Glioblastoma cellular architectures are predicted through the characterization of two-cell interactions

Significance Microscopic analysis of cellular architectures within a diseased tissue often provides an independent assay relative to measurements of molecular biomarkers from that same tissue. Both methods may point to the same disease state, but the relationship between the two is often not clear. We explore a connection by investigating how growth factor-driven protein signaling depends upon the distance separating pairs of cancer cells. A thermodynamic-derived theory identifies the intercellular separation that corresponds to the steady state of the signaling. That length scale is found to be the dominant cell separation distance in bulk tissue culture. The approach is tested in one cell line and validated in another, and may provide insight into the diffusive nature of certain brain cancer phenotypes. To understand how pairwise cellular interactions influence cellular architectures, we measured the levels of functional proteins associated with EGF receptor (EGFR) signaling in pairs of U87EGFR variant III oncogene receptor cells (U87EGFRvIII) at varying cell separations. Using a thermodynamics-derived approach we analyzed the cell-separation dependence of the signaling stability, and identified that the stable steady state of EGFR signaling exists when two U87EGFRvIII cells are separated by 80–100 μm. This distance range was verified as the characteristic intercellular separation within bulk cell cultures. EGFR protein network signaling coordination for the U87EGFRvIII system was lowest at the stable state and most similar to isolated cell signaling. Measurements of cultures of less tumorigenic U87PTEN cells were then used to correctly predict that stable EGFR signaling occurs for those cells at smaller cell–cell separations. The intimate relationship between functional protein levels and cellular architectures explains the scattered nature of U87EGFRvIII cells relative to U87PTEN cells in glioblastoma multiforme tumors.

Anomalous Features of EMT during Keratinocyte Transformation

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike “conventional EMT”, these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

On a fundamental structure of gene networks in living cells.

Computers are organized into hardware and software. Using a theoretical approach to identify patterns in gene expression in a variety of species, organs, and cell types, we found that biological systems similarly are comprised of a relatively unchanging hardware-like gene pattern. Orthogonal patterns of software-like transcripts vary greatly, even among tumors of the same type from different individuals. Two distinguishable classes could be identified within the hardware-like component: those transcripts that are highly expressed and stable and an adaptable subset with lower expression that respond to external stimuli. Importantly, we demonstrate that this structure is conserved across organisms. Deletions of transcripts from the highly stable core are predicted to result in cell mortality. The approach provides a conceptual thermodynamic-like framework for the analysis of gene-expression levels and networks and their variations in diseased cells.

Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

Oncogenic transformation is a complex, multistep process, which goes through several stages before complete malignant transformation occurs. To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, papillomavirus transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and papillomavirus-transformed keratinocytes from early (E) and late (L) passages and from benzo[a]pyrene-treated L cells (BP). The transformed cells exhibit similar transcriptional changes to clinical cervical carcinoma. The number of transcripts expressed progressively decreased during the evolution from K to BP cells. Bioinformatic analysis, validated by detailed biochemical analysis, revealed substantial contraction of both pro- and antiapoptotic networks during transformation. Nonetheless, L and BP cells were not resistant to apoptotic stimuli. At doses of cisplatin that led to 30-60% apoptosis of K and E cells, transformed L and BP cells underwent 80% necrotic cell death, which became the default response to genotoxic stress. Moreover, appreciable necrotic fractions were observed in the cervical carcinoma cell line, HeLa, in response to comparable doses of cisplatin. The shrinkage of biochemical networks, including the apoptotic network, may allow a cancer cell to economize on energy usage to facilitate enhanced proliferation but leaves it vulnerable to stress. This study supports the hypothesis that the process of cancer transformation may be accompanied by a shift from apoptosis to necrosis.

Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation.

Up-regulation of AMP-activated Protein Kinase in Cancer Cell Lines Is Mediated through c-Src Activation

We report that the activation level of AMP-dependent protein kinase AMPK is elevated in cancer cell lines as a hallmark of their transformed state. In OVCAR3 and A431 cells, c-Src signals through protein kinase Cα, phospholipase Cγ, and LKB1 to AMPK. AMPK controls internal ribosome entry site (IRES) dependent translation in these cells. We suggest that AMPK activation via PKC might be a general mechanism to regulate IRES-dependent translation in cancer cells.

Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement

Significance We demonstrate the role of free energy in determining the direction of motion in a biological process. A thermodynamics-motivated approach is used to show that free-energy changes in cell–cell signaling determine the force gradient for directed cell motion. Using isolated cell pairs and assays of secreted protein levels, we compute the free energy of the cell–cell signaling network as a function of cell–cell separation. The changes in free energy as a function of separation correspond to a potential-energy gradient that can influence cell–cell motion. Recordings of cell motion trajectories were compared with the direction of the gradient. Neutralizing the secreted proteins most involved in establishing the free-energy gradient cancels the directed motion. Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell–cell signaling can depend on cell–cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell–cell signaling that induces a directed cell–cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell–cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell–cell distances. We show that glioblastoma cell–cell movement can be described as Brownian motion biased by cell–cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

Heterogeneity of Gene Expression in Murine Squamous Cell Carcinoma Development—The Same Tumor by Different Means

Transformation is a complex process, involving many changes in the cell. In this work, we investigated the transcriptional changes that arose during the development of squamous cell carcinoma (SCC) in mice. Using microarray analysis, we looked at gene expression during different stages in cancer progression in 31 mice. By analyzing tumor progression in each mouse separately, we were able to define the global changes that were common to all 31 mice, as well as significant changes that occurred in fewer individuals. We found that different genes can contribute to the tumorigenic process in different mice, and that there are many ways to acquire the malignant properties defined by Hanahan and Weinberg as “hallmarks of cancer”. Eventually, however, all these changes lead to a very similar cancerous phenotype. The finding that gene expression is strongly heterogeneous in tumors that were induced by a standardized protocol in closely related mice underscores the need for molecular characterization of human tumors and personalized therapy.