Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463∕624 and 460∕605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.cell polarity | intravital imaging | Keima | two-photon microscopy | mKate

Here, we used quantitative proteomics analysis to identify novel therapeutic targets in cancer stem cells and/or progenitor cells. For this purpose, mammospheres from two ER-positive breast cancer cell lines (MCF7 and T47D) were grown in suspension using low-attachment plates and directly compared to attached monolayer cells grown in parallel. This allowed us to identify a subset of proteins that were selectively over-expressed in mammospheres, relative to epithelial monolayers. We focused on mitochondrial proteins, as they appeared to be highly upregulated in both MCF7 and T47D mammospheres. Key mitochondrial-related enzymes involved in beta-oxidation and ketone metabolism were significantly upregulated in mammospheres, as well as proteins involved in mitochondrial biogenesis, and specific protein inhibitors of autophagy/mitophagy. Overall, we identified >40 “metabolic targets” that were commonly upregulated in both MCF7 and T47D mammospheres. Most of these “metabolic targets” were also transcriptionally upregulated in human breast cancer cells in vivo, validating their clinical relevance. Based on this analysis, we propose that increased mitochondrial biogenesis and decreased mitochondrial degradation could provide a novel mechanism for the accumulation of mitochondrial mass in cancer stem cells. To functionally validate our observations, we utilized a specific MCT1/2 inhibitor (AR-C155858), which blocks the cellular uptake of two types of mitochondrial fuels, namely ketone bodies and L-lactate. Our results indicate that inhibition of MCT1/2 function effectively reduces mammosphere formation, with an IC-50 of ~1 μM, in both ER-positive and ER-negative breast cancer cell lines. Very similar results were obtained with oligomycin A, an inhibitor of the mitochondrial ATP synthase. Thus, the proliferative clonal expansion of cancer stem cells appears to require oxidative mitochondrial metabolism, related to the re-use of monocarboxylic acids, such as ketones or L-lactate. Our findings have important clinical implications for exploiting mitochondrial metabolism to eradicate cancer stem cells and to prevent recurrence, metastasis and drug resistance in cancer patients. Importantly, a related MCT1/2 inhibitor (AZD3965) is currently in phase I clinical trials in patients with advanced cancers: http://clinicaltrials.gov/show/NCT01791595.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20 -30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the ␤-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrinlinked kinase (ILK), binds to the cytoplasmic domain of ␤ 1 and ␤ 3 integrin subunits and promotes anchorageindependent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide ؊54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3␤ (GSK-3␤) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.The cyclin D1 gene encodes a regulatory subunit of a serinethreonine kinase that phosphorylates and inactivates the tumor suppressor pRB (1). The abundance of cyclin D1 was shown to be rate-limiting in cellular proliferation induced by diverse signaling pathways in fibroblasts and breast epithelial cells, including MCF7 cells (2, 3). Homozygous deletion of the cyclin D1 gene in mice results in defects in mammary gland development (4, 5) and in serum-induced cellular proliferation (6). The abundance of cyclin D1 is increased in more than 30% of human breast tumors, and overexpression of cyclin D1 under control of the MMTV 1 promoter in transgenic mice induces mammary adenocarcinoma (7). The majority of breast cancer cell lines and mammary tumors induced by transgenic overexpression of either pp60 v-src or ErbB-2 oncogenes overexpress cyclin D1, suggesting that the induction of cyclin D1 may play an important role in mammary tumorigenesis (8). The cyclin D1 gene is activated by mitogenic stimuli induced by G-protein-

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR␥ induces hepatic steatosis, and liganded PPAR␥ promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR␥ function, transactivation, expression, and promoter activity. PPAR␥ transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB-and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix ( The cyclin-dependent kinase holoenzymes are a family of serine/threonine kinases that play a pivotal role in controlling progression through the cell cycle (38,47). Dysregulation of the cell cycle control apparatus is an almost uniform aberration in tumorigenesis (48). The cyclins encode regulatory subunits of the kinases which phosphorylate specific proteins, including the retinoblastoma (pRB) protein, to promote transition through specific cell cycle checkpoints (47, 57). Cyclin D1 plays a pivotal role in G 1 /S phase cell cycle progression in fibroblasts and is rate limiting in growth factor-or estrogen-induced mammary epithelial cell proliferation (29, 67). Cyclin D1 overexpression is found in Ͼ30% of human breast cancers, correlating with poor prognosis (23). Several different oncogenic signals induce cyclin D1 expression, including mutations of the Ras and Wnt/APC/␤-catenin pathway (2, 49). Mammary-targeted expression of cyclin D1 is sufficient for the induction of mammary adenocarcinoma, and cyclin D1 Ϫ/Ϫ mice are resistant to ErbB2-induced tumorigenesis (53,64).In addition to binding cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) and pRB, cyclin D1 forms physical associations with P/CAF (p300/CBP-associated factor), Myb, MyoD, and the cyclin D1 myb-like binding protein (DMP1) (16,20,31,39). Binding of cyclin D1 to the estrogen receptor alpha (ER␣) enhances ligand-independent reporter gene activity, and liganded androgen receptor reporter gene activity is inhibited by cyclin D1 (33, 39, 68). The in vivo or genetic evidence indicating a requirement for cyclin D1 in nuclear receptor function remained to be determined. The peroxisome proliferator-activator receptors, including PPAR␣, PPAR␥, and PPAR␦, are ligand-activated nuclear receptors (42). Their modular structure resembles those of other nuclear hormone receptors with N-terminal AF-1, a DNA binding domain, and a carboxyl-terminal ligand-binding domain (LBD). PPAR␥ was cloned as a transcription factor involved in fat cell differentiation and is required for the induction of adipocyte differentiation (41, 51). Adenoviral delivery of PPAR␥ to the livers of mice induces hepatic steatosis, consistent with an important role for PPAR␥ in hepatocellular lipid biosynthesis (65). The PPAR␥ ligands include eicosanoids, such as 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ 2 ), and synthetic ligands of the thiazolidinedione (TZD) class. PPAR␥ ...

N-cadherin is up-regulated in aggressive breast carcinomas, but its mechanism of action in vivo remains unknown. Transgenic mice coexpressing N-cadherin and polyomavirus middle T antigen (PyVmT) in the mammary epithelium displayed increased pulmonary metastasis, with no differences in tumor onset or growth relative to control PyVmT mice. PyVmT-N-cadherin tumors contained higher levels of phosphorylated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) than PyVmT controls, and phosphorylated ERK staining was further increased in pulmonary metastases. Tumor cell isolates from PyVmT-N-cadherin mice exhibited enhanced ERK activation, motility, invasion, and matrix metalloproteinase-9 (MMP-9) expression relative to PyVmT controls. MAPK/ERK kinase 1 inhibition in PyVmT-N-cadherin cells reduced MMP-9 production and invasion but not motility. Furthermore, inactivation of fibroblast growth factor receptor in PyVmT-N-cadherin cells reduced motility, invasion, and ERK activation but had no effect on PyVmT cells. Thus, de novo expression of N-cadherin in mammary ducts enhances metastasis of breast tumors via enhanced ERK signaling. [Cancer Res 2007;67(7):3106-16]

Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H₂O₂) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients.

The Wnt/␤-catenin/Tcf and IB/NF-B cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/␤-catenin signaling occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear ␤-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IB kinase complex (IKK) that phosphorylates IB contains IKK␣, IKK␤, and IKK␥. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/␤-catenin and IB pathways in mitogenic signaling. Mitogenic induction of G 1 -S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1 Ϫ/Ϫ cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IB/IKK␣ or ␤-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKK␣. In IKK␣ Ϫ/Ϫ cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKK␣ restored normal mitogen induction of cyclin D1 through a Tcf site. In IKK␣ Ϫ/Ϫ cells, ␤-catenin phosphorylation was decreased and purified IKK␣ was sufficient for phosphorylation of ␤-catenin through its N-terminus in vitro. Because IKK␣ but not IKK␤ induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKK␣ and IKK␤ may alter their substrate and signaling specificities to regulate mitogeninduced DNA synthesis through distinct mechanisms. INTRODUCTIONThe Wingless/Wnt pathway plays a crucial role in development and cell cycle control (Cadigan and Nusse, 1997;Huelsken and Behrens, 2000). Dysregulation of the Wingless/ (Wnt)/␤-catenin/Tcf pathway has been implicated in tumorigenesis of diverse types (Polakis, 2000a). Axin/Conductin, together with APC, promote ␤-catenin degradation through serine-threonine phosphorylation of the ␤-catenin N-terminus by GSK3␤, which targets ␤-catenin for ubiquitination by a SCF ␤-TRCP (␤-transducin repeat-containing protein) ubiquitin ligase complex (Fuchs et al., 1999;Winston et al., 1999) and its degradation by the proteasome. On induction of Wnt signaling by extracellular ligands, the Frizzled receptors are activated. The activity of GSK3␤ and its effect on ␤-catenin is antagonized by Dishevelled, a downstream target of Frizzled, thus preventing the degradation of ␤-cate- nin by the proteasome. The resulting accumulation of ␤-catenin leads to its nuclear translocation and binding to Tcf/Lef transcription factors to induce target genes including cyclin D1 and c-Myc (He et al., 1998;Shtutman et al., 1999;Huelsken and Behrens, 2000). In addition to components in the Wnt signaling pathway, several other pathways can regulate ␤-catenin/Tcf signaling and gene expression and confer aberrant cellular growth. The protein encoded by Gas6, a growth factor of the vitamin K-dependent family, which binds member...

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