Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38alpha mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38alpha positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38alpha controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38alpha leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-Ras(G12V)-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38alpha has a key role in the regulation of lung cell renewal and tumorigenesis.

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.

Loss of thyroid hormone receptors (TR) is a common feature in some tumors, although their role in tumor progression is currently unknown. We show here that expression of TRB1 in hepatocarcinoma and breast cancer cells reduces tumor growth, causes partial mesenchymal-to-epithelial cell transition, and has a striking inhibitory effect on invasiveness, extravasation, and metastasis formation in mice. In cultured cells, TRB1 abolishes anchorage-independent growth and migration, blocks responses to epidermal growth factor, insulin-like growth factor-I, and transforming growth factor B, and regulates expression of genes that play a key role in tumorigenicity and metastatic growth. The receptor disrupts the mitogenic action of growth factors by suppressing activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling pathways that are crucial for cell proliferation and invasiveness. Furthermore, increased aggressiveness of skin tumors is found in genetically modified mice lacking TRs, further demonstrating the role of these receptors as inhibitors of tumor progression. These results define a novel role for the thyroid hormone receptor as a metastasis suppressor gene, providing a starting point for the development of novel therapeutic strategies for the treatment of human cancer. [Cancer Res 2009;69(2):501-9]

Corepressors are involved in gene silencing by various transcriptional repressor proteins such as MAD/MAX and MxiI (2), YY1 (74), KRAB domain proteins (27), NGF1-A, KROX 20 (59, 63) and some members of the nuclear hormone receptor (NHR) superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR) (8,19,38,50). Both TR and RAR repress gene activity in the absence of hormone in vivo (3, 4, 21, 73) and in vitro (28, 67, 68). This repression is mediated by a silencing domain in the carboxy terminus, encompassing about 250 amino acids (aa) (3,33,46,58). In addition to the silencing function, TR and RAR harbor several other functions C-terminal to their DNA binding domain (DBD) including dimerization, hormone binding and hormone-dependent transactivation. These activities can be transferred to heterologous proteins and therefore represent functional domains (for reviews, see references 6, 50, and 65).Gene silencing by NHRs is relieved by addition of the cognate ligand, which induces a conformational change and transforms the receptor into a transcriptional activator. In this way, both hormone binding and the small conserved receptor activation domain, AF2/AF2-AD/4/c (8, 11, 12, 22, 49, 50), representing helix 12 (14, 38, 55, 57, 71), are required to dissociate corepressors from the receptors (8,9,19,38). It is yet unknown why so many different coactivators are involved in transcriptional activation by NHRs. Gene silencing by TR, RAR, Rev-erbA␣, and COUP-TF is mediated, at least in part, by corepressors in vivo (8, 10, 19, 25, 38, 61) and in vitro (68), which bind to the unliganded (apo) receptors. Only one class of nuclear receptor corepressors has been identified, which exhibit hormone-sensitive interaction. This class contains two related members, 38). These corepressors were isolated by the yeast twohybrid system and bind to the silencing domains of TR and RAR only in the absence of ligand. Hormone binding by the receptor leads to dissociation of these corepressors. Furthermore, SMRT and N-CoR are localized in the cell nucleus and harbor an autonomous silencing function when bound to DNA (19,38). The mechanism of repression by the SMRT/N-CoR class involves interaction with SIN3 and a histone deacetylase function (1,35,52).Here, we describe a novel corepressor, Alien, which is unrelated to SMRT and N-CoR and is highly conserved from humans to Drosophila. Conserved sequences are even found in Ricinus communis and Caenorhabditis elegans. Alien interacts with TR only in the absence of hormone and does not interact with RAR, retinoid X receptor (RXR), or glucocorticoid

Purpose: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/b-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance.Experimental Design: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/b-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors.Results: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear b-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear b-catenin content and progressed earlier upon PI3K/AKT/ mTOR inhibition. Nuclear b-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not.Conclusions: High nuclear b-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear b-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/b-catenin inhibition and together with b-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/b-catenin inhibitors.

Nearly 50% of human malignancies exhibit unregulated RAS-ERK signaling; inhibiting it is a valid strategy for antineoplastic intervention. Upon activation, ERK dimerize, which is essential for ERK extranuclear, but not for nuclear, signaling. Here, we describe a small molecule inhibitor for ERK dimerization that, without affecting ERK phosphorylation, forestalls tumorigenesis driven by RAS-ERK pathway oncogenes. This compound is unaffected by resistance mechanisms that hamper classical RAS-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two understudied concepts in cancer therapy: (1) the blockade of sub-localization-specific sub-signals, rather than total signals, as a means of impeding oncogenic RAS-ERK signaling and (2) targeting regulatory protein-protein interactions, rather than catalytic activities, as an approach for producing effective antitumor agents.

The RAS to extracellular signal-regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention.

Purpose: Within the aim of advancing precision oncology, we have generated a collection of patientderived xenografts (PDX) characterized at the molecular level, and a preclinical model of colon cancer metastasis to evaluate drug-response and tumor progression.Experimental Design: We derived cells from 32 primary colorectal carcinomas and eight liver metastases and generated PDX annotated for their clinical data, gene expression, mutational, and histopathological traits. Six models were injected orthotopically into the cecum wall of NOD-SCID mice in order to evaluate metastasis. Three of them were treated with chemotherapy (oxaliplatin) and three with API2 to target AKT activity. Tumor growth and metastasis progression were analyzed by positron emission tomography (PET).Results: Patient-derived cells generated tumor xenografts that recapitulated the same histopathological and genetic features as the original patients' carcinomas. We show an 87.5% tumor take rate that is one of the highest described for implanted cells derived from colorectal cancer patients. Cecal injection generated primary carcinomas and distant metastases. Oxaliplatin treatment prevented metastasis and API2 reduced tumor growth as evaluated by PET.Conclusions: Our improved protocol for cancer cell engraftment has allowed us to build a rapidly expanding collection of colorectal PDX, annotated for their clinical data, gene expression, mutational, and histopathological statuses. We have also established a mouse model for metastatic colon cancer with patient-derived cells in order to monitor tumor growth, metastasis evolution, and response to treatment by PET. Our PDX models could become the best preclinical approach through which to validate new biomarkers or investigate the metastatic potential and drug-response of individual patients.