Dataset Information

MCF7 SILAC Comparison of Protein Expression for Acidosis Biomarkers

ABSTRACT: MCF7 cells were grown in culture under low pH and physiological pH conditions in order to generate candidate biomarkers for tumor acidosis. SILAC was used to enable comparison of protein expression levels using relative quantification. Two biological replicates were prepared using "label flipping," and each fraction was analyzed in duplicate by LC-MS/MS. Sample nomenclature includes N for "normal" or physiological pH versus L for low pH as the first letter with H for "heavy" and L for "light" as the second letter. Therefore, one experiment is described as NH-LL, while the other is NL-LH. Fifteen IEF fractions were generated and analyzed with LC-MS/MS (RSLC and Orbitrap, Thermo) for each SILAC pair .

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: John Koomen

LAB HEAD: Robert Gillies

PROVIDER: PXD002860 | Pride | 2020-07-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	062012_Gillies_MCF7_SILAC_NH_LL_01.RAW	Raw
	062012_Gillies_MCF7_SILAC_NH_LL_01_120703073656.RAW	Raw
	062012_Gillies_MCF7_SILAC_NH_LL_02.RAW	Raw
	062012_Gillies_MCF7_SILAC_NH_LL_02_120703124815.RAW	Raw
	062012_Gillies_MCF7_SILAC_NH_LL_03.RAW	Raw

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Publications

Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.

Damaghi Mehdi M Tafreshi Narges K NK Lloyd Mark C MC Sprung Robert R Estrella Veronica V Wojtkowiak Jonathan W JW Morse David L DL Koomen John M JM Bui Marilyn M MM Gatenby Robert A RA Gillies Robert J RJ

Nature communications 20151210

Early cancers are avascular and hence, profoundly acidic. Pre-malignant cells must adapt to acidosis to thrive in this hostile microenvironment. Here, we investigate MCF-7 cells that are adapted to grow in acidic conditions using SILAC proteomics and we reveal a significant upregulation of lysosomal proteins. Prominent among these is LAMP2 that functions to protect lysosomal membranes from acid proteolysis. LAMP2 upregulation by acidosis is confirmed both in vitro and in vivo. Furthermore, we sh ...[more]

PMID: 26658462

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Project description:Lactic acidosis and hypoxia are two prominent tumor microenvironmental stresses that are both known to exert important influences on gene expression and phenotypes of cancer cells. But very little is known about the cross-talk and interaction between these two stresses. We performed gene expression analysis of MCF7 cells exposed to lactic acidosis, hypoxia and combined lactic acidosis and hypoxia. We found the hypoxia response elicited under hypoxia was mostly abolished upon simultaneous exposure to lactic acidosis. The repression effects are due to loss of HIF-1Î± protein synthesis under lactic acidosis. In addition, we showed lactic acidosis strongly synergizes with hypoxia to activate the unfold protein response (UPR) and inflammation response which are highly similar to amino acid deprivation responses (AAR). The statistical factor analysis of hypoxia and lactic acidosis responses indicated that ATF4 locus, an important activator in the UPR/AAR pathway, is amplified in subsets of breast tumors and cancer cell lines. Varying ATF4 levels dramatically affect the ability to survive the post-stress recovery from hypoxia and lactic acidosis and may suggest its selection of ATF4 amplification in human cancers. These data suggest that lactic acidosis interacts with hypoxia by both inhibiting the canonical hypoxia response and while activating the UPR and inflammation response. Gain of ATF4 locus may offer survival advantages to allow successful adaptation to frequent fluctuations of oxygen and acidity in tumor microenvironment. Collectively, our studies have provided linkage between the short-term transcriptional responses to the long term selection of the DNA copy number alterations (CNAs) under tumor microenvironmental stresses. RNAs from MCF7 cells exposed to control condition (ambient air ~21% O2, no lactate and neutral pH), lactic acidosis (ambient air, 10 mM Lactate and pH 6.7), hypoxia (1% pO2, no lactate and neutral pH) and the combined lactic acidosis and hypoxia (1% pO2, 10 mM Lactate and pH 6.7) condition for 24 hours were extracted by miRVana kits (Ambion) and hybridized to Affymetrix Human genome 133A 2.0 arrays with standard protocol.