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The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1. Significance: This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin–mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin–binding sites in the intron of Rnf43. This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor–expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562–75. ©2017 AACR.

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial–mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426–37. ©2013 AACR.

The FoxM1 transcription factor gene is overexpressed in cancer. Its expression is stimulated by oncogenic signaling pathways and reactive oxygen species. It is also a target of regulation by the tumor suppressor genes. The transcriptional activity of FoxM1 depends upon activation by cyclin and cyclin-dependent kinases as well as Plk1. FoxM1 stimulates expression of several genes involved in the cell cycle progression. Moreover, it supports proliferation of tumor cells by stimulating expression of the antioxidant genes and reducing oxidative stress. A new study provides evidence that FoxM1, in the absence of its inhibitor, the tumor suppressor Arf, drives metastasis of hepatocellular carcinoma (HCC). It induces an epithelial–mesenchymal–like transition phenotype in HCC cells, increases cell migration, and induces premetastatic niche at the distal organ of metastasis. FoxM1 directly activates genes involved in multiple steps of metastasis. In this review, we discuss the evidence for a master regulatory role of FoxM1 in tumor metastasis. Cancer Res; 71(13); 4329–33. ©2011 AACR.

Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state. Cancer Res; 71(12); 4292–302. ©2011 AACR.

Inherent and acquired therapeutic resistance in breast cancer remains a major clinical challenge. In human breast cancer samples, overexpression of the oncogenic transcription factor FoxM1 has been suggested to be a marker of poor prognosis. In this study, we report that FoxM1 overexpression confers resistance to the human epidermal growth factor receptor 2 monoclonal antibody Herceptin and microtubule-stabilizing drug paclitaxel, both as single agents and in combination. FoxM1 altered microtubule dynamics to protect tumor cells from paclitaxel-induced apoptosis. Mechanistic investigations revealed that the tubulin-destabilizing protein Stathmin, whose expression also confers resistance to paclitaxel, is a direct transcriptional target of FoxM1. Significantly, attenuating FoxM1 expression by small interfering RNA or an alternate reading frame (ARF)–derived peptide inhibitor increased therapeutic sensitivity. Our findings indicate that targeting FoxM1 could relieve therapeutic resistance in breast cancer. Cancer Res; 70(12); 5054–63. ©2010 AACR.

The regulation of androgen receptor (AR) expression in prostate cancer is still poorly understood. The activation of the epidermal growth factor receptor (EGFR) in prostate cancer cells was previously shown to lower AR expression by a rapamycin-sensitive, posttranscriptional mechanism involving the AR mRNA 5′-untranslated region (5′-UTR). In a search for an intermediate within the EGFR/phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway that regulates AR at this site, we identified the nucleic acid–binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNP-K), by mass spectrometric analysis of Akt immune complexes from lipid raft–enriched subcellular fractions. We show here that hnRNP-K is a novel inhibitor of AR mRNA translation that regulates androgen-responsive gene expression and prostate cancer cell proliferation. A functional hnRNP-K binding site involved in down-regulating AR protein levels was identified in the AR mRNA 5′-UTR. Further analysis revealed that hnRNP-K is also able to inhibit AR translation in the absence of the 5′-UTR, consistent with the presence of additional predicted hnRNP-K binding sites within the AR open reading frame and in the 3′-UTR. Immunohistochemical analysis of a human prostate cancer tissue microarray revealed an inverse correlation between hnRNP-K expression and AR protein levels in organ-confined prostate tumors and a substantial decline in cytoplasmic hnRNP-K in metastases, despite an overall increase in hnRNP-K levels in metastatic tumors. These data suggest that translational inhibition of AR by hnRNP-K may occur in organ-confined tumors but possibly at a reduced level in metastases. HnRNP-K is the first protein identified that directly interacts with and regulates the AR translational apparatus. [Cancer Res 2009;69(6):2210–8]

Colon cancer is one of the deadliest cancers worldwide because of its metastasis to other essential organs. Metastasis of colon cancer involves a complex set of events, including epithelial-to-mesenchymal transition (EMT) that increases invasiveness of the tumor cells. Here, we show that the xeroderma pigmentosum group E (XPE) gene product, damaged DNA-binding protein (DDB)-2, is downregulated in high-grade colon cancers, and it plays a dominant role in the suppression of EMT of the colon cancer cells. Depletion of DDB2 promotes mesenchymal phenotype, whereas expression of DDB2 promotes epithelial phenotype. DDB2 constitutively represses genes that are the key activators of EMT, indicating that DDB2 is a master regulator of EMT of the colon cancer cells. Moreover, we observed evidence that DDB2 functions as a barrier for EMT induced by hypoxia and TGF-β. Also, we provide evidence that DDB2 inhibits metastasis of colon cancer. The results presented here identify a transcriptional regulatory pathway of DDB2 that is directly linked to the mechanisms that suppress metastasis of colon cancer. Cancer Res; 73(12); 3771–82. ©2013 AACR.

Introduction: The BCR-ABL1 tyrosine kinase induces malignant transformation of B cells at the pre-B cell checkpoint and induces Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). In a genome-wide search, we identified FOXM1 as a transcription factor that is specifically upregulated at the pre-B cell checkpoint. FOXM1 is a forkhead box transcription factor and a key regulator of cell growth by promoting cell cycle progression. Results: We transformed murine pre-B cells with a retroviral BCR-ABL1 expression vector and observed upregulation of Foxm1 protein levels. Consistent with this, FOXM1 protein levels in patient-derived Ph+ ALL samples are ∼10-fold higher than in healthy B cells and B cell precursors (n=5; P=0.01). In a cohort of 83 Ph+ ALL patients, the FOXM1 promoter region was significantly de-methylated compared to normal pre-B cells (n=12; P=2.4x10e-7). In order to evaluate a potential predictive value of FOXM1 expression in ALL cells, we correlated FOXM1 mRNA levels with clinical outcome. High FOXM1 expression correlates with shorter overall survival in patients with ALL (n=12, P=0.05) and was predictive of a higher risk of relapse (n=21; P=7.3x10e-5). To study the functional role of FOXM1 in Ph+ ALL, we developed a genetic model for Foxm1 loss-of-function using Foxm1fl/fl mice. Cre-mediated, inducible deletion of Foxm1 decreases ALL cell viability (n=3; P=0.001) and reduced proliferative capacity (n=3; S-phase Foxm1+/+ 59% vs 42% for Foxm1-/-; P=0.0004). The ability to form colonies in vitro was significantly decreased by deletion of Foxm1 (n=3, P=8.9x10e-6). Injection of 1x10e5 Foxm1+/+ or Foxm1-/- leukemic cells into NOD/SCID mice revealed reduced leukemogenesis in vivo (n=7; P=0.0035). In contrast, normal B cell precursors were unaffected by Foxm1 deletion both, in vitro and in vivo. In addition, Foxm1-deleted ALL cells revealed a strikingly higher sensitivity towards Imatinib in dose-response curves compared to control cells (IC50=420 nM for Foxm1+/+ vs 160 nM for Foxm1-/-). Mechanistically, we found that FOXM1 decreased levels of reactive oxygen species via upregulation of the antioxidant response molecule catalase. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide (AA 26-44) and the natural occurring antibiotic Thiostrepton. Both bind and inhibit the function of FOXM1, and induced apoptosis in Ph+ ALL. Patient-derived Ph+ ALL cells were xenografted into NOD/SCID recipients and then treated with Thiostrepton i.v. for 7 consecutive days. Thiostrepton-treatment resulted in significantly prolonged overall survival (29 vs 35 days; n=7; P=0.0014) and reduced leukemia burden as measured by luciferase bioimaging, as compared to vehicle controls. Conclusion: Taken together, our data identify the pre-B cell-specific transcription factor FOXM1 as a novel therapeutic target for the treatment of TKI-resistant Ph+ ALL. Citation Format: Maike V. Buchner, Eugene Park, Lars Klemm, Huimin Geng, Dragana Kopanja, Pradip Raychaudhuri, Markus Müschen. Identification of FOXM1 as therapeutic target in Philadelphia chromosome-positive acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2014-484

Forkhead box transcription factor FoxM1 is overexpressed in many types of human cancers and promotes tumor development in multiple ways. It is also considered as a potential target for cancer therapy. Because greater than half of the human cancers harbor p53 mutations, we investigate the effect of FoxM1-targeting in spontaneous p53-null tumors. We show that reducing FoxM1 inhibits growth of the p53 null thymic lymphoma and sarcoma cells. In addition, ablation of FoxM1 retards the growth of the p53-null tumors in mouse xenograft model. Moreover, induction of apoptotic cell death is observed in p53 null cells after deletion of FoxM1, accompanied by decreased expression of the FoxM1 target gene Survivin. The ARF26-44 peptide, which inhibits the activity of FoxM1 by targeting it to the nucleolus, is able to effectively induce apoptosis in the p53 null spontaneous tumors. Together, our observations suggest that FoxM1 functions are critical for p53-null tumors and FoxM1 is a therapeutic target of human tumors carrying loss of function p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1858. doi:1538-7445.AM2012-1858

Invasion and metastasis are the hallmarks of malignant tumor progression and the principal cause of death related to solid tumors. Epithelial to mesenchymal transition (EMT) is intricately associated with cancer progression, as it confers cancer cells the ability to invade and metastasize to a distant organ. Here we identify a novel function of nucleotide excision repair protein DDB2 as an important antagonist of the EMT in colon carcinoma through its maintenance of epithelial phenotype. In human colon carcinoma samples, loss of DDB2 expression is highly correlated with the aggressiveness of the disease. Moreover, stimuli (TGF-beta and hypoxia) that induce EMT inhibit expression of DDB2; and loss of DDB2 is sufficient to promote EMT of colon cancer cells. The mechanism involves DDB2 mediated transcriptional repression of VEGF and MMP3. DDB2 represses these genes by histone deacetylation. We also observed that loss of DDB2 results in resistance to anoikis, an important barrier to metastasis. The lack of anoikis response is associated with increased Akt and ERK activity. Furthermore, treatment with Phenethyl Isothiocyanate (PEITC), a promising cancer chemopreventive component, augmented E-cadherin expression that is related to an increase in DDB2 expression. The present study suggests that anti-metastatic effect of PEITC involves important role of DDB2. Together, our results show that loss of DDB2 expression is both necessary and sufficient for EMT during colon carcinoma progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2410. doi:1538-7445.AM2012-2410

Over-expression of the Forkhead box M1 (FoxM1) transcription factor coincides with highly aggressive, poorly differentiated hepatocellular carcinoma (HCC). Over-expression of FoxM1 in HCC also correlates with poorer overall survival after hepatectomy. Since HCC progression involves hepatic cancer stem cells (HCSCs), we considered the roles of FoxM1 in HCSCs. We found that FoxM1 expression correlates with the expression of the stemness genes in human HCC cell lines. Interestingly, depletion of FoxM1 in human HCC lines causes a selective reduction in the population of the CD90+ HCSCs due to selective apoptosis. Moreover, FoxM1 silencing decreases sphere formation potential of the same cell lines, suggesting that FoxM1 is critical for survival of the HCSCs. Consistent with the loss of HCSCs, depletion of FoxM1 causes decrease in tumorigenic potential of the HCC cells in vitro as well as inhibition of tumor formation in mouse xenografts. Furthermore, we found that FoxM1 directly regulates expression of CD44, previously shown to be important for the tumorigenic potential of CD90+ HCC cells. These studies have exciting implications in further considerations of FoxM1 as a target of HCC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3479. doi:1538-7445.AM2012-3479

Robert Henry (Rob) Costa, an authority on liver biology and cancer, died on September 1, 2006 at his home in Oak Park, IL. ![Graphic][1] Rob was born in Galesburg, IL and grew up in Bethesda, MD. He attended the University of California at Irvine, where he received his doctorate in Molecular