Tumor cells, with stem-like properties, are highly aggressive and often display drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung, and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumor xenografts or in clinical specimens from lung cancer patients that had progressed on erlotinib. Mechanistically, αvβ3, in the unligated state, recruits KRAS and RalB to the tumor cell plasma membrane, leading to the activation of TBK-1/NFκB. In fact, αvβ3 expression and the resulting KRAS/RalB/NFκB pathway were both necessary and sufficient for tumor initiation, anchorage-independence, self-renewal, and erlotinib resistance. Pharmacological targeting of this pathway with Bortezomib reversed both tumor stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but they reveal a therapeutic strategy to sensitize such tumors to RTK inhibition.

Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications. However, growing and processing large quantities of undifferentiated hESCs is a challenging task. Conventionally, hESCs are passaged as clusters, which can limit their growth efficiency and use in downstream applications. This study demonstrates that hESCs can be passaged as single cells using Accutase, a formulated mixture of digestive enzymes. In contrast to trypsin treatment, Accutase treatment does not significantly affect the viability and proliferation rate of hESC dissociation into single cells. Accutase-dissociated single cells can be separated by FACS and proliferate as fully pluripotent hESCs. An Oct4-eGFP reporter construct engineered into hESCs was used to monitor the pluripotency of hESCs passaged with Accutase. Compared to collagenase-passaged hESCs, Accutase-treated cultures contained a larger proportion of undifferentiated (Oct4-positive) cells. Additionally, Accutase-passaged undifferentiated hESCs could be grown as monolayers without the need for monitoring and/or selection for quality hESC colonies.

The ErbB2 (Neu) receptor tyrosine kinase is frequently overexpressed in human breast cancers, and this phenotype correlates with a poor clinical prognosis. We examined the effects of the mammalian target of rapamycin inhibitor, rapamycin, on mammary tumorigenesis in transgenic mice bearing an activated ErbB2 (NeuYD) transgene in the absence or presence of a second transgene encoding vascular endothelial growth factor (VEGF). Treatment of NeuYD or NeuYD Â VEGF mice with rapamycin dramatically inhibited tumor growth accompanied by a marked decrease in tumor vascularization. Two key events that may underlie the antitumor activity of rapamycin were decreased expression of ErbB3 and inhibition of hypoxia-inducible factor-1-dependent responses to hypoxic stress. Rapamycin exposure caused only a modest inhibition of the proliferation of tumor-derived cell lines in standard monolayer cultures, but dramatically inhibited the growth of the same cells in three-dimensional cultures, due in part to the induction of apoptotic cell death. These studies underscore the therapeutic potential of mammalian target of rapamycin inhibitors in ErbB2-positive breast cancers and indicate that, relative to monolayer cultures, three-dimensional cell cultures are more predictive in vitro models for studies of the antitumor mechanisms of rapamycin and related compounds. (Cancer Res 2005; 65(12): 5325-36)

Maternal Embryonic Leucine zipper Kinase (MELK) is expressed in several developing tissues, in the adult germ line, and in adult neural progenitors. MELK expression is elevated in aggressive undifferentiated tumors correlating with poor patient outcome in human breast cancer. To investigate the role of MELK in mammary tumorigenesis in vivo we used a MELK-GFP reporter mouse, which allows prospective isolation of MELK expressing cells based on GFP fluorescence. We found that in the normal mammary gland, cells expressing high levels of MELK were enriched for proliferating cells, expressing markers of mammary progenitors. The isolation of cells with high levels of MELK in mammary tumors from MMTV-Wnt1/MELK-GFP bitransgenic mice resulted in a significant enrichment of tumorsphere formation in culture and tumor initiation after transplantation into mammary fat pads of syngeneic mice. Furthermore, using lentiviral delivery of MELK-specific shRNA and limiting dilution cell transplantations we demonstrated that MELK function is required for mammary tumorigenesis in vivo. Our findings identify MELK as potential target in breast tumor initiating cells.

The Ets2 transcription factor is regulated by mitogen-activated protein (MAP) kinase phosphorylation of a single threonine residue. We generated by gene targeting a single codon mutation in Ets2 substituting Ala for the critical Thr-72 phosphorylation site (Ets2 A72 Most cancers are epithelial in origin. However, a significant and sometimes predominate portion of breast, colon, stomach, and pancreas tumors are host fibroblasts, endothelial cells, inflammatory cells, smooth muscle cells, adipocytes, and extracellular matrix known collectively as stroma (6). Much less is known about the molecular genetics of tumor-stroma interaction than is known about epithelial transformation. Tissue stroma can both suppress and promote tumor progression (16,57). Among the examples of stroma-neoplasia communication is the role of matrix metalloproteinases produced by fibroblasts and invasive inflammatory cells in the angiogenic switch associated with pancreatic and skin neoplastic progression (5, 10).)Ets2 is one of over 25 transcription factors in the human genome that utilize the Ets winged helix-loop-helix DNA-binding domain (30, 54). Extracellular signal-regulated kinase 2 (Erk2) binds to Ets2 through a docking site in the evolutionarily conserved, N-terminal pointed domain of a subset of Ets transcription factors (44). The signal transduction pathway from growth factor receptors, such as fibroblast growth factor (FGF) receptor and epidermal growth factor receptor (EGFR; Erbb1), through the Ras-mitogen-activated protein kinase pathway to the pointed domain of Ets transcription factors is used for regulating developmental transitions in Drosophila melanogaster and Caenorhabditis elegans and growth factor signaling in humans and mice. In Drosophila the closest relative of Ets2, the Pointed-P2 product of the pointed gene, is key in Ras-pathway-dependent photoreceptor development (40) and in tracheal ductal morphogenesis (42). In mammals, Ets2 has been implicated in growth factor stimulation of macrophages (7,31,45,49); FGF stimulation (59); and oncogenic stimulation by Neu (23), Ras (60), and Raf (36). The expression of either wild-type Ets2 or a dominant-negative form of Ets2 can reverse the transformed characteristics of human or mouse cancer cell lines (17, 18). Inactivation of Ets2 by a gene-targeted deletion of the DNA-binding domain and nuclear localization signal (Ets2 db1 ) results in embryonic lethality associated with deficient expression of matrix metalloproteinase 9 (MMP-9) and CD31 in trophoblastic derivatives (59). Haploinsufficiency of Ets2 is sufficient to restrict mammary tumors arising from polyomavirus middle T (PyMT) oncogene expression driven by the mouse mammary tumor virus (MMTV) long terminal repeat promoter (38).Here, we show that mutation of Thr-72 creates a hypomorphic allele of Ets2 (Ets2 A72 ), which acts, at least in part, in host stroma to restrict mammary tumors. This tumor restriction is genetically downstream of vascular endothelial growth factor (VEGF) production and is correlated with decrea...

No abstract

SUMMARY While integrin αvβ3 expression is linked to breast cancer progression its role in epithelial development is unclear. Here, we show that αvβ3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvβ3 is a luminal progenitor marker in the virgin gland, we noted increased αvβ3 expression in MaSCs at mid-pregnancy. Accordingly, mice lacking αvβ3 or expressing a signaling deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity and expression of Slug, a master regulator of MaSCs. Surprisingly, αvβ3 deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. TGFβ2 induced αvβ3 expression leading to Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells αvβ3 was necessary and sufficient for Slug activation, tumorsphere formation and tumor initiation. Thus, pregnancy-associated MaSCs require a TGFβ2/αvβ3/Slug pathway, which may contribute to breast cancer progression and stemness.

Identifying the molecular basis for cancer cell dependence on oncogenes such as KRAS can provide new opportunities to target these addictions. Here, we identify a novel role for the carbohydrate-binding protein Galectin-3 as a lynchpin for KRAS dependence. By directly binding to the cell surface receptor integrin αvβ3, Galectin-3 gives rise to KRAS addiction by enabling multiple functions of KRAS in anchorage-independent cells, including formation of macropinosomes that facilitate nutrient uptake and ability to maintain redox balance. Disrupting αvβ3/Galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvβ3-expressing KRAS mutant lung and pancreatic cancer patient-derived xenografts and spontaneous tumors in mice. Our work reveals Galectin-3 as a druggable target for KRAS-addicted lung and pancreas cancer, and indicates integrin αvβ3 as a biomarker to identify susceptible tumors.