Subpopulation Of Slow-cycling Cells Research Articles

The glutathione S-transferase Gstt1 drives survival and dissemination in metastases.

Identifying the adaptive mechanisms of metastatic cancer cells remains an elusive question in the treatment of metastatic disease, particularly in pancreatic cancer (pancreatic adenocarcinoma, PDA). A loss-of-function shRNA targeted screen in metastatic-derived cells identified Gstt1, a member of the glutathione S-transferase superfamily, as uniquely required for dissemination and metastasis, but dispensable for primary tumour growth. Gstt1 is expressed in latent disseminated tumour cells (DTCs), is retained within a subpopulation of slow-cycling cells within existing metastases, and its inhibition leads to complete regression of macrometastatic tumours. This distinct Gstt1high population is highly metastatic and retains slow-cycling phenotypes, epithelial-mesenchymal transition features and DTC characteristics compared to the Gstt1low population. Mechanistic studies indicate that in this subset of cancer cells, Gstt1 maintains metastases by binding and glutathione-modifying intracellular fibronectin, in turn promoting its secretion and deposition into the metastatic microenvironment. We identified Gstt1 as a mediator of metastasis, highlighting the importance of heterogeneity and its influence on the metastatic tumour microenvironment.

Abstract PR011: The glutathione S-transferase, Gstt1 is a robust driver of survival and dissemination in metastases

Abstract Identifying adaptive mechanisms of metastatic cancer cells remains an elusive question in the treatment of metastatic disease, particularly in pancreatic cancer (PDA), where the majority of patients present with metastatic lesions at the time of diagnosis. A loss-of-function shRNA targeted screen in metastatic-derived cells identified Gstt1, a member of the glutathione S-transferase superfamily, as uniquely required for metastasis and dissemination however dispensable for primary tumor growth. Gstt1 is expressed in early disseminated tumor cells (DTCs), is retained within a subpopulation of slow-cycling cells within established metastases and its inhibition completely abolished macrometastatic lesions. This distinct Gstt1high population is highly metastatic and retains slow-cycling phenotypes, EMT features, and DTC characteristics compared to the Gstt1low population. Mechanistic studies indicate that in this subset of cells, Gstt1 maintains metastases by binding to and modifying intracellular fibronectin, regulating fibronectin secretion from cancer cells and deposition into the metastatic microenvironment. We identified Gstt1 as a novel mediator of metastasis, highlighting the importance of metastatic heterogeneity and its influence on the metastatic tumor microenvironment. Citation Format: Christina M. Ferrer. The glutathione S-transferase, Gstt1 is a robust driver of survival and dissemination in metastases [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR011.

Cancer Stem Cells and the Slow Cycling Phenotype: How to Cut the Gordian Knot Driving Resistance to Therapy in Melanoma.

Simple SummaryCancer stem cells play a central role in the development of cancer and are poorly sensitive to standard chemotherapy and radiotherapy. Furthermore, they are also responsible for the onset of drug resistance. This also occurs in malignant melanoma, the deadliest form of skin cancer. Hence, cancer stem cells eradication is one of the main challenges for medical oncology. Here, we conducted a bioinformatics approach aimed to identify the main circuits and proteins underpinning cancer stem cell fitness in melanoma. Several lessons emerged from our work and may help to conceptualize future therapeutic approaches to prolong the efficacy of current therapies.Cancer stem cells (CSCs) have historically been defined as slow cycling elements that are able to differentiate into mature cells but without dedifferentiation in the opposite direction. Thanks to advances in genomic and non-genomic technologies, the CSC theory has more recently been reconsidered in a dynamic manner according to a “phenotype switching” plastic model. Transcriptional reprogramming rewires this plasticity and enables heterogeneous tumors to influence cancer progression and to adapt themselves to drug exposure by selecting a subpopulation of slow cycling cells, similar in nature to the originally defined CSCs. This model has been conceptualized for malignant melanoma tailored to explain resistance to target therapies. Here, we conducted a bioinformatics analysis of available data directed to the identification of the molecular pathways sustaining slow cycling melanoma stem cells. Using this approach, we identified a signature of 25 genes that were assigned to four major clusters, namely (1) kinases and metabolic changes, (2) melanoma-associated proteins, (3) Hippo pathway and (4) slow cycling/CSCs factors. Furthermore, we show how a protein−protein interaction network may be the main driver of these melanoma cell subpopulations. Finally, mining The Cancer Genome Atlas (TCGA) data we evaluated the expression levels of this signature in the four melanoma mutational subtypes. The concomitant alteration of these genes correlates with the worst overall survival (OS) for melanoma patients harboring BRAF-mutations. All together these results underscore the potentiality to target this signature to selectively kill CSCs and to achieve disease control in melanoma.

PO-263 Epigenetic modulation of cell metabolism and its effects on cell survival in melanoma

Introduction Melanoma is an aggressive tumour with a median survival of only 6–12 month after occurrence of metastasis. In detail, one key factor of drug resistance mechanism in melanoma pathogenesis is cell heterogeneity. A defined cell subpopulation of slow cycling cells marked by a high histone demethylase JARID1B expression are therapy resistant and are most likely responsible for tumour repopulation. Moreover, cells with a slow cycling phenotype are described to modulate metabolism under targeted therapy. Therefore, the aim of my thesis is to characterise metabolic ‘hubs’ within metabolic landscaping analysis of melanoma cells. Material and methods We induced JARID1Blow and JARID1Bhigh phenotypes in WM3734 melanoma cells via shRNA-mediated knockdown and Doxycyclin-inducible Tet3G-Overexpression and prepared for a subpopulation-specific LC-HRMS based metabolomics landscaping analysis. Additionally, we investigated if chemical H3K4 demethylase inhibition could be used to specifically target the JARID1B-driven malignant metabolic phenotype in melanoma cells. Results and discussions JARID1Bhigh expressing melanoma cells show upregulated metabolites of glutathione-redox-system and pentose phosphate pathway. Moreover, several upregulated metabolites in JARID1Bhigh melanoma cells confirm existing data stating that those tumour cells do not follow the Warburg effect, but rather depend on mitochondrial OXPHOS to gain ATP and maintain a multi-resistant phenotype. Furthermore, a treatment with H3K4 demethylase inhibitors modulates the detected active metabolic pathways in melanoma cells functionally and also impairs cell proliferation and invasion capacity. Conclusion Our present metabolic landscaping analysis unravelled heterogeneous metabolic profiles in melanoma pointing to a possible relevance for therapy resistance of distinct melanoma cell subpopulations. In detail, we could profile the metabolome of multi-drug resistant JARID1Bhigh melanoma cells including their underlying enzymatic hubs. To overcome drug resistance in future, such hubs could be either directly targeted or indirectly by inhibiting their epigenetic regulators.

PO-264 Mitochondrial metabolism: a key factor of myeloid leukemic cell response upon exposure to tyrosine kinase inhibitors

IntroductionMelanoma is an aggressive tumour with a median survival of only 6–12 month after occurrence of metastasis. In detail, one key factor of drug resistance mechanism in melanoma pathogenesis is cell heterogeneity. A defined cell subpopulation of slow cycling cells marked by a high histone demethylase JARID1B expression are therapy resistant and are most likely responsible for tumour repopulation. Moreover, cells with a slow cycling phenotype are described to modulate metabolism under targeted therapy. Therefore, the aim of my thesis is to characterise metabolic ‘hubs’ within metabolic landscaping analysis of melanoma cells.Material and methodsWe induced JARID1Blow and JARID1Bhigh phenotypes in WM3734 melanoma cells via shRNA-mediated knockdown and Doxycyclin-inducible Tet3G-Overexpression and prepared for a subpopulation-specific LC-HRMS based metabolomics landscaping analysis. Additionally, we investigated if chemical H3K4 demethylase inhibition could be used to specifically target the JARID1B-driven malignant metabolic phenotype in melanoma cells. PO-263 Figure 1Results and discussionsJARID1Bhigh expressing melanoma cells show upregulated metabolites of glutathione-redox-system and pentose phosphate pathway. Moreover, several upregulated metabolites in JARID1Bhigh melanoma cells confirm existing data stating that those tumour cells do not follow the Warburg effect, but rather depend on mitochondrial OXPHOS to gain ATP and maintain a multi-resistant phenotype. Furthermore, a treatment with H3K4 demethylase inhibitors modulates the detected active metabolic pathways in melanoma cells functionally and also impairs cell proliferation and invasion capacity.ConclusionOur present metabolic landscaping analysis unravelled heterogeneous metabolic profiles in melanoma pointing to a possible relevance for therapy resistance of distinct melanoma cell subpopulations. In detail, we could profile the metabolome of multi-drug resistant JARID1Bhigh melanoma cells including their underlying enzymatic hubs. To overcome drug resistance in future, such hubs could be either directly targeted or indirectly by inhibiting their epigenetic regulators.

Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma

Evidence suggests that multiple tumors, including pancreatic adenocarcinoma, display heterogeneity in parameters that are critical for tumor formation, progression and metastasis. Understanding heterogeneity in solid tumors is increasingly providing a plethora of new diagnostic and therapeutic approaches. In this study, a particular focus was put on identifying a subpopulation of stem cell-like, slow cycling tumor cells in a pancreas adenocarcinoma cell lines. Using a label retention technique a subpopulation of slow cycling cells (DiI+/SCC) was identified and further evaluated in the BxPC-3 and Panc03.27 cell lines. These slowly cycling cells managed to retain the lipophilic labeling dye DiI, while the bulk of the cells (>94%) did not. The DiI+/SCC population, showed only a partial overlap with the CSC markers CD24+/CD44+, CD133+ and ALDH but they survived chemotherapeutic treatment, and were able to recreate the initial heterogeneous tumor cell population. DiI+/SCCs exhibited an increased invasive potential as compared with their non-label retaining, faster cycling cells (DiI−/FCC). They also had increased tumorigenic potential and morphological changes resembling cells that have undergone an epithelial to mesenchymal transition (EMT). Analysis of DiI+/SCC cells by real time PCR revealed a selective up-regulation of tell tale components of the Hedgehog/TGFβ pathways, as well as a down-regulation of EGFR, combined with a shift in crucial components implied in EMT. The presented findings offer an expanded mechanistic understanding that associates tumor initiating potential with cycling speed and EMT in pancreatic cancer cell lines.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-009-9260-0) contains supplementary material, which is available to authorized users.