Final Report Summary - MITOCANCERSTEM (Role of Mitochondrial Physiology in Tumor Stem Cell Resistance to Chemotherapeutics)
Project context
Although advances in cancer research have heightened cases of long-term survival, tumour regrowth is still a major concern. A subpopulation of stem cells has been identified in many types of cancer, and the ability of these cells to evade treatment is a strong mechanism for tumour regrowth. Our hypothesis is that the differential physiology of mitochondria in cancer stem cells vs. differentiated cells results in a different susceptibility to chemotherapeutics.
Project objectives
The aim of our Marie Curie project is to study several aspects of mitochondria and programmed cell death in cancer stem cells (CSCs) with particular interest in how chemotherapeutic drugs act in these type cells. Another side of our proposal is to test different agents which are or can be used as potential chemotherapeutics in order to establish their effectiveness against tumour stem cells. The project has trained the researcher in several aspects of mitochondrial/cell biology - in addition, this a powerful tool for finding mitochondrial poisons that could be used in patients. P19 embryonal carcinoma cells are a unique system on the basis of which cancer stem-like cells can be compared, after treating with 1microM retinoic acid, with more differentiated cancer cells (dCCs) in the same cell line to investigate differences that could explain the resistance of CSCs and to suggest novel drug targets.
Project results
Our results showed a resistance of CSCs to chemotherapeutic poisons such as etoposide, dichloroacetate and rapamycin. CSCs indicated the highest protein levels in the pluripotency markers Oct 3/4, Sox-2 and Nanog. Meanwhile, dCCs had an increase in differentiation markers Troma-1, Musashi and neuron-specific betaIII-tubulin. Mitochondrial remodelling during CSC to dCC transition was characterised by a conversion from small round bodies to long polarised filaments, although no alterations were observed in mtDNA copy number, COXIV and Tom20 expression used as markers of mitochondrial mass. In accordance with morphological results, mitochondrial function and dynamics differ between groups, dCCs presented the highest mitochondrial respiration, fusion processes, and ATP and ROS production.
Of particular importance was the increase in adenine nucleotide translocator expression, in calpain activity and in lysosomal cathepsins activities observed in dCCs. Furthermore, CSCs showed a strong antioxidant protection with higher content in mitochondrial superoxide dismutase and p53. However, there were no differences in protein oxidative damage between both types of tumour cells. Active caspase-3-cleaved fragment was detected in CSCs, although caspase 3, 8 and 9-like activities were higher in dCCs. Some markers of autophagy, such as cathepsin D/B ratio, DRAM and beclin-1, were found in CSCs. However, the low level of p62 observed in dCCs indicates a normal autophagic response and flux, whereas the increased level of p62 together with the lack in lysosomal proteases activities in CSCs could indicate a block in the flux, resulting from an inhibition of the pathway. Although autophagy plays a key role in controlling and triggering cell-death processes, it may represent a cell survival strategy which promotes an additional system for maintenance of mitochondrial quality and homeostasis during stem cell differentiation.
Project outcome
This Marie Curie project allowed us to contribute more effectively to international efforts to combat major diseases such as cancer which is a leading cause of death worldwide. Thus, uncovering differences between tumour stem cells and differentiated cells as regards chemotherapeutic effects may lead to a better understanding of why CSCs can survive treatments, and potentially identify novel targets for new therapeutic approaches which eliminate CSCs. The literature is somewhat scarce concerning the role of mitochondria in the susceptibility of tumour stem cells to chemotherapeutics. Based on specific differences regarding mitochondrial physiology and mechanisms of programmed cell death, more directed, effective and selective therapeutics can be used in oncology. Our work reveals that quiescent mitochondria together with high antioxidant protection, high p53 expression and low calpain and cathepsins activities may contribute to maintaining genetic stability and resistance to apoptotic stimuli in CSCs. Therefore, we suggest that stem-line therapies must include the stimulation of mitochondrial and lysosomal activities to activate the autophagy-apoptosis switch, increasing the sensitivity of CSCs to cell death.
Although advances in cancer research have heightened cases of long-term survival, tumour regrowth is still a major concern. A subpopulation of stem cells has been identified in many types of cancer, and the ability of these cells to evade treatment is a strong mechanism for tumour regrowth. Our hypothesis is that the differential physiology of mitochondria in cancer stem cells vs. differentiated cells results in a different susceptibility to chemotherapeutics.
Project objectives
The aim of our Marie Curie project is to study several aspects of mitochondria and programmed cell death in cancer stem cells (CSCs) with particular interest in how chemotherapeutic drugs act in these type cells. Another side of our proposal is to test different agents which are or can be used as potential chemotherapeutics in order to establish their effectiveness against tumour stem cells. The project has trained the researcher in several aspects of mitochondrial/cell biology - in addition, this a powerful tool for finding mitochondrial poisons that could be used in patients. P19 embryonal carcinoma cells are a unique system on the basis of which cancer stem-like cells can be compared, after treating with 1microM retinoic acid, with more differentiated cancer cells (dCCs) in the same cell line to investigate differences that could explain the resistance of CSCs and to suggest novel drug targets.
Project results
Our results showed a resistance of CSCs to chemotherapeutic poisons such as etoposide, dichloroacetate and rapamycin. CSCs indicated the highest protein levels in the pluripotency markers Oct 3/4, Sox-2 and Nanog. Meanwhile, dCCs had an increase in differentiation markers Troma-1, Musashi and neuron-specific betaIII-tubulin. Mitochondrial remodelling during CSC to dCC transition was characterised by a conversion from small round bodies to long polarised filaments, although no alterations were observed in mtDNA copy number, COXIV and Tom20 expression used as markers of mitochondrial mass. In accordance with morphological results, mitochondrial function and dynamics differ between groups, dCCs presented the highest mitochondrial respiration, fusion processes, and ATP and ROS production.
Of particular importance was the increase in adenine nucleotide translocator expression, in calpain activity and in lysosomal cathepsins activities observed in dCCs. Furthermore, CSCs showed a strong antioxidant protection with higher content in mitochondrial superoxide dismutase and p53. However, there were no differences in protein oxidative damage between both types of tumour cells. Active caspase-3-cleaved fragment was detected in CSCs, although caspase 3, 8 and 9-like activities were higher in dCCs. Some markers of autophagy, such as cathepsin D/B ratio, DRAM and beclin-1, were found in CSCs. However, the low level of p62 observed in dCCs indicates a normal autophagic response and flux, whereas the increased level of p62 together with the lack in lysosomal proteases activities in CSCs could indicate a block in the flux, resulting from an inhibition of the pathway. Although autophagy plays a key role in controlling and triggering cell-death processes, it may represent a cell survival strategy which promotes an additional system for maintenance of mitochondrial quality and homeostasis during stem cell differentiation.
Project outcome
This Marie Curie project allowed us to contribute more effectively to international efforts to combat major diseases such as cancer which is a leading cause of death worldwide. Thus, uncovering differences between tumour stem cells and differentiated cells as regards chemotherapeutic effects may lead to a better understanding of why CSCs can survive treatments, and potentially identify novel targets for new therapeutic approaches which eliminate CSCs. The literature is somewhat scarce concerning the role of mitochondria in the susceptibility of tumour stem cells to chemotherapeutics. Based on specific differences regarding mitochondrial physiology and mechanisms of programmed cell death, more directed, effective and selective therapeutics can be used in oncology. Our work reveals that quiescent mitochondria together with high antioxidant protection, high p53 expression and low calpain and cathepsins activities may contribute to maintaining genetic stability and resistance to apoptotic stimuli in CSCs. Therefore, we suggest that stem-line therapies must include the stimulation of mitochondrial and lysosomal activities to activate the autophagy-apoptosis switch, increasing the sensitivity of CSCs to cell death.