Background Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. Methods The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Results Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. Conclusions The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell ...

Cells and organisms have developed homeostatic mechanisms which protect them against a changing environment. How growth and homeostasis interact is still not well understood, but of increasing interest to the molecular and synthetic biology community to recognize and design control circuits which can oppose the diluting effects of cell growth. In this paper we describe the performance of selected negative feedback controllers in response to different applied growth laws and time dependent outflow perturbations of a controlled variable. The approach taken here is based on deterministic mass action kinetics assuming that cell content is instantaneously mixed. All controllers behave ideal in the sense that they for step-wise perturbations in volume and a controlled compound A are able to drive A precisely back to the controllers’ theoretical set-points. The applied growth kinetics reflect experimentally observed growth laws, which range from surface to volume ratio growth to linear and exponential growth. Our results show that the kinetic implementation of integral control and the structure of the negative feedback loop are two properties which affect controller performance. Best performance is observed for controllers based on derepression kinetics and controllers with an autocatalytic implementation of integral control. Both are able to defend exponential growth and perturbations, although the autocatalytic controller shows an offset from its theoretical set-point. Controllers with activating signaling using zero-order or bimolecular (antithetic) kinetics for integral control behave very similar but less well. Their performance can be improved by implementing negative feedback structures having repression/derepression steps or by increasing controller aggressiveness. Our results provide a guide what type of feedback structures and integral control kinetics are suitable to oppose the dilution effects by different growth laws and time dependent perturbations on a deterministic level.

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Most cancer cells rely on aerobic glycolysis and increased glucose uptake for the production of biosynthetic precursors needed to support rapid proliferation. Increased glucose uptake and glycolytic activity may result in intracellular acidosis and increase of osmotically active substances, leading to cell swelling. This causes dilution of cellular constituents, which can markedly influence cellular reactions and the function of proteins, and hence, control mechanisms used by cancer cells to maintain a highly glycolytic phenotype must be robust to dilution. In this paper, we review the literature on cancer cell metabolism and glucose uptake, and employ mathematical modeling to examine control mechanisms in cancer cell metabolism that show robust homeostatic control in the presence of dilution. Using differential gene expression data from the Expression Atlas database, we identify the key components of glucose uptake in cancer, in order to guide the construction of a mathematical model. By simulations of this model we show that while negative feedback from downstream glycolytic metabolites to glucose transporters is sufficient for homeostatic control of glycolysis in a constant cellular volume, it is necessary to control intermediate glycolytic enzymes in order to achieve homeostatic control during growth. With a focus on glucose uptake in cancer, we demonstrate a systems biology approach to the identification, reduction, and analysis of complex regulatory systems. SIGNIFICANCE Rapid proliferation and increased glycolytic activity in cancer cells lead to dilution of cellular constituents, which can markedly influence cellular reactions and the function of proteins. Therefore, control mechanisms used by cancer cells to maintain a highly glycolytic phenotype must be robust to dilution. We construct a mathematical model of glucose uptake in cancer, and using a systems biology approach to the analysis of regulatory networks, identify the presence of integral control motifs as a means for achieving dilution resistance. Furthermore, we show that while negative feedback from downstream glycolytic metabolites to glucose transporters is sufficient for homeostatic control of glycolysis in a constant cellular volume, it is necessary to control intermediate glycolytic enzymes to achieve homeostatic control during growth.

Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring based on non-genomic adaptation was validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of tumor suppressors and negative MAPK regulators, dual specific phosphatases, reengages mitogenic signaling. Upregulation of growth factors or cytokine receptors triggers signaling pathways circumventing BRAF blockage. Changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, an upregulation of pigmentation in inhibitor resistant melanoma cells was observed. Cellular pathways utilized during inhibitor resistance promoted melanogenesis, a pathway which partially overlaps with MAPK signaling. Upstream regulator analysis suggested gene expression changes of forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy resistant cancer.