Huihui Li1, Matjaz Rokavec1, Longchang Jiang1, David Horst2, Heiko Hermeking3. 1. Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Germany. 2. Institute of Pathology, Ludwig-Maximilians-University Munich, Germany; German Cancer Consortium (DKTK), Partner Site Munich, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany. 3. Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Germany; German Cancer Consortium (DKTK), Partner Site Munich, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address: heiko.hermeking@med.uni-muenchen.de.
Abstract
BACKGROUND & AIMS: In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a selective advantage under conditions of hypoxia, but little is known about the mediators of this effect. METHODS: Isogenic colorectal cancer (CRC) cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called INH3, HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 human colon cancers. We analyzed data on human colorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of epithelial-to-mesenchymal transition (EMT), or MIR34A or was associated with metastasis. We determined the effects of disruption Mir34a, Mir34b, and Mir34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging. RESULTS: The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34A gene in p53-defective CRC cells, whereas expression of MIR34A was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34A was required for hypoxia-induced EMT, invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34A. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34A reduced activation of STAT3 in p53-deficient CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-deficient CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption of Mir34a, Mir34b, and Mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with TP53 mutations and metastasis to the liver. CONCLUSIONS: HIF1A represses, whereas p53 increases, expression of MIR34A in CRC cells. MIR34A reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia.
BACKGROUND & AIMS: In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a selective advantage under conditions of hypoxia, but little is known about the mediators of this effect. METHODS: Isogenic colorectal cancer (CRC) cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called INH3, HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 humancolon cancers. We analyzed data on humancolorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of epithelial-to-mesenchymal transition (EMT), or MIR34A or was associated with metastasis. We determined the effects of disruption Mir34a, Mir34b, and Mir34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging. RESULTS: The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34A gene in p53-defective CRC cells, whereas expression of MIR34A was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34A was required for hypoxia-induced EMT, invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34A. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34A reduced activation of STAT3 in p53-deficient CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-deficient CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption of Mir34a, Mir34b, and Mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with TP53 mutations and metastasis to the liver. CONCLUSIONS:HIF1A represses, whereas p53 increases, expression of MIR34A in CRC cells. MIR34A reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia.
Authors: Yan Zhao; Xuedong Kang; Alexander Barsegian; Jian He; Alejandra Guzman; Ryan P Lau; Reshma Biniwale; Madhuri Wadhra; Brian Reemtsen; Meena Garg; Nancy Halnon; Fabiola Quintero-Rivera; Wayne W Grody; Glen Van Arsdell; Stanley F Nelson; Marlin Touma Journal: J Mol Med (Berl) Date: 2020-06-12 Impact factor: 4.599