While metabolic defects have been investigated extensively in differentiated tumor cells, much less attention has been directed to the metabolic properties of stem-like cells that repopulate tumors (tumor-repopulating cells [TRC]). Here we show that melanoma TRCs cultured in 3D soft fibrin gels reprogram glucose metabolism by hijacking the cytosolic enzyme phosphoenolpyruvate carboxykinase (PCK1), a key player in gluconeogenesis. Surprisingly, upregulated PCK1 in TRCs did not mediate gluconeogenesis but promoted glucose side-branch metabolism, including in the serine and glycerol-3-phosphate pathways. Moreover, this retrograde glucose carbon flow strengthened rather than antagonized glycolysis and glucose consumption. Silencing PCK1 or inhibiting its enzymatic activity slowed the growth of TRCs in vitro and impeded tumorigenesis in vivo. Overall, our work unveiled metabolic features of tumor-repopulating cells in melanoma that have implications for targeting a unique aspect of this disease.

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2–PI3K and ERBB–RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2–PI3K and ERBB–RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN,% and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies. Cancer Res; 75(8); 1–7. ©2015 AACR.

Development of targeted therapeutics required translationally-relevant preclinical models with well characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2-PI3K and ERBB-RAS pathways and response to cetuximab. PDX molecular data was compared to that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2-PI3K and ERBB-RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in non-hypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histological diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8/52) of PDXs and response strictly restricted to KRAS wild type models. The response rate reaches 53% (8/15) when KRAS, BRAF and NRAS mutations are concomitantly excluded, proving a functional cross validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies.

FOXP3 functions not only as the master regulator in regulatory T cells, but also as an X-linked tumor suppressor. The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor-suppressive activity remains largely unknown. Using chromatin immunoprecipitation (ChIP) sequencing, we identified a series of potential FOXP3-targeted miRNAs in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulates NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In ChIP assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146–NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contributes to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3–miR-146–NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects. Cancer Res; 75(8); 1–11. ©2015 AACR.

FOXP3 functions not only as the master regulator in regulatory T cells but also as an X-linked tumor suppressor. The tumor suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor suppressive activity remains largely unknown. Using chromatin immunoprecipitation sequencing, we identified a series of potential FOXP3-targeted microRNAs (miRs) in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulate NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In chromatin immunoprecipitation assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146-NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contribute to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3-miR-146-NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects.