Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity, but its expression is increased in some cancers via stabilization with HSP90-associated chaperones. Here, we show that MIF stabilization is tumor-specific in an acute colitis-associated colorectal cancer (CRC) mouse model, leading to tumor-specific functions and selective therapeutic vulnerabilities. Therefore, we demonstrate that a Mif deletion reduced CRC tumor growth. Further, we define a dual role for MIF in CRC tumor progression. Mif deletion protects mice from inflammation-associated tumor initiation, confirming the action of MIF on host inflammatory pathways; however, macrophage recruitment, neoangiogenesis, and proliferative responses are reduced in Mif-deficient tumors once the tumors are established. Thus, during neoplastic transformation, the function of MIF switches from a proinflammatory cytokine to an angiogenesis promoting factor within our experimental model. Mechanistically, Mif-containing tumor cells regulate angiogenic gene expression via a MIF/CD74/MAPK axis in vitro. Clinical correlation studies of CRC patients show the shortest overall survival for patients with high MIF levels in combination with CD74 expression. Pharmacological inhibition of HSP90 to reduce MIF levels decreased tumor growth in vivo, and selectively reduced the growth of organoids derived from murine and human tumors without affecting organoids derived from healthy epithelial cells. Therefore, novel, clinically relevant Hsp90 inhibitors provide therapeutic selectivity by interfering with tumorigenic MIF in tumor epithelial cells but not in normal cells. Furthermore, Mif-depleted colonic tumor organoids showed growth defects compared to wild-type organoids and were less susceptible toward HSP90 inhibitor treatment. Our data support that tumor-specific stabilization of MIF promotes CRC progression and allows MIF to become a potential and selective therapeutic target in CRC.

The vast majority of human tumors with p53 mutations undergo loss of the remaining wildtype p53 allele (loss-of-heterozygosity, p53LOH). p53LOH has watershed significance in promoting tumor progression. However, driving forces for p53LOH are poorly understood. Here we identify the repressive WTp53–HSF1 axis as one driver of p53LOH. We find that the WTp53 allele in AOM/DSS chemically-induced colorectal tumors (CRC) of p53^R248Q/+ mice retains partial activity and represses heat-shock factor 1 (HSF1), the master regulator of the proteotoxic stress response (HSR) that is ubiquitously activated in cancer. HSR is critical for stabilizing oncogenic proteins including mutp53. WTp53-retaining CRC tumors, tumor-derived organoids and human CRC cells all suppress the tumor-promoting HSF1 program. Mechanistically, retained WTp53 activates CDKN1A/p21, causing cell cycle inhibition and suppression of E2F target MLK3. MLK3 links cell cycle with the MAPK stress pathway to activate the HSR response. In p53^R248Q/+ tumors WTp53 activation by constitutive stress represses MLK3, thereby weakening the MAPK-HSF1 response necessary for tumor survival. This creates selection pressure for p53LOH which eliminates the repressive WTp53-MAPK-HSF1 axis and unleashes tumor-promoting HSF1 functions, inducing mutp53 stabilization enabling invasion.

A prerequisite for gain-of-function (GOF) p53 missense mutants (mutp53) is protein stabilization. Moreover, a prerequisite for mutp53 stabilization is loss of the remaining wildtype (WT) p53 allele (loss-of-heterozygosity, p53LOH) in mutp53/+ tumors. Thus, GOF, mutp53 stabilization and p53LOH are strictly linked. However, the driving force for p53LOH is unknown. Typically, heterozygous tumors are an instable transition state. Here we identify the repressive WTp53-HSF1 axis as the driver of p53LOH. We find that the WTp53 allele in AOM/DSS-induced colorectal tumors (CRC) of p53 ^R248Q/+ mice retains its haploid transcriptional activity. Notably, WTp53 represses heat-shock factor 1 (HSF1) activity, the master transcription factor of the proteotoxic stress defense response (HSR) that is ubiquitously and constitutively activated in cancer tissues. HSR is critical for stabilizing oncogenic proteins including mutp53. WTp53-retaining murine CRC tumors and tumor-derived organoids and human CRC cells all suppress the tumor-promoting HSF1 transcriptional program. Mechanistically, the retained WTp53 allele activates CDKN1A/p21, leading to cell cycle inhibition and suppression of the E2F target gene MLK3. MLK3 links cell cycle to the MAPK stress pathway to activate the HSR response. We show that in p53 ^R248Q/+ tumors WTp53 activation by constitutive stress (emanating from proliferative/metabolic stresses and genomic instability) represses MLK3, consequently inactivating the MAPK-HSF1 response necessary to ensure tumor survival. This creates strong selection pressure for p53LOH which eliminates the repressive WTp53-HSF1 axis and unleashes the tumor-promoting HSF1 functions, inducing mutp53 stabilization and enabling invasion. HIGHLIGHTS: heterozygous p53 ^R248Q/+ tumors retain p53 transcriptional activity in a mouse model of colorectal cancer (CRC) wildtype p53 actively represses the tumor-promoting HSF1-regulated chaperone system and proteotoxic stress response the repressive WTp53 – HSF1 axis creates a selective pressure for WTp53 loss-of-heterozygosity in CRC tumors p53 loss-of-heterozygosity enables stabilization of the gain-of-function p53 ^R248Q mutant protein which in turn enables CRC invasion