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Breast cancer is the most frequently diagnosed cancer and the leading cause of death by cancer among females worldwide. An overwhelming majority of these deaths is because of metastasis. Estrogen stimulates and promotes growth of breast tumors, whereas transforming growth factor-beta (TGF-β) signaling promotes invasion and metastasis. We previously reported that estrogen and estrogen receptor alpha (ERα) suppressed breast cancer metastasis by inhibiting TGF-β signaling, whereas antiestrogens that suppress breast cancer growth, such as the selective ER modulator tamoxifen (TAM) or the pure antiestrogen fulvestrant (ICI 182,780), cannot suppress TGF-β signaling or breast cancer invasiveness. Therefore, we predicted that a compound that inhibits TGF-β signaling but does not facilitate ERα signaling would be ideal for suppressing breast cancer invasiveness and growth. In the present study, we identified an ideal candidate compound, N-23. Like estrogen, N-23 strongly decreased expression of TGF-β/Smad target gene plasminogen activator inhibitor-1 (PAI-1), but it did not increase the expression of ERα target gene pS2. While estrogen decreased the levels of phosphorylated Smad2 and Smad3, N-23 had no effect. In addition, TGF-β-dependent recruitment of Smad3 to the PAI-1 gene promoter was inhibited in the presence of estrogen or N-23. We also investigated the effects of N-23 on proliferation, migration, and invasion of breast cancer cells. In contrast to estrogen, N-23 inhibited the cellular proliferation of breast cancer cells. Moreover, we showed that N-23 suppressed the migration and invasion of breast cancer cells to the same extent as by estrogen. Taken together, our findings indicate that N-23 may be a candidate compound that is effective in inhibiting breast cancer progression.

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-β promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-β signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-β signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-β signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-β receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-β target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression. (Cancer Sci 2011; 102: 1501–1508)

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

Background: Colorectal cancer and Mammary analogue secretory carcinoma harboring Neurotrophic tyrosine receptor kinase (NTRK/TRK) gene rearrangements developed resistance to Entrectinib, ALK/ROS/TRK inhibitor. We previously demonstrated that the next generation pan-TRK inhibitor,ONO-5390556 may potently overcome Entrectinib-resistance mutations. Furthermore, inhibition of Trk phosphorylation in tumors has excellent correlation with the in vivo anti-tumor effect by ONO-5390556 (AACR2016, Kozaki et al). Purpose: Accurate measurement of target inhibition in a phase 1 clinical trial is critical to informing selection of appropriate doses for ONO-5390556 in more advanced clinical trials. Plasma inhibitory activity (PIA) assay has recently been performed as a surrogate assay in clinical trials of FLT3, MET and ALK kinase inhibitors. The assay employs the incubation of Tyrosine Kinase expressing cell lines in aliquots of plasma collected at various time points from patients treated with TKI. Herein, we report the preclinical evaluation of PIA assay in accordance with an inhibition of TRK phosphorylation in tumors. Methods: KM12 cells, human colorectal cancer cell lines expressing TPM3-TRKA, were implanted subcutaneously into nude mice. Mice were randomized when the mean tumor volume was 150-600 mm3. Tumors and blood were collected from mice, 2, 4, 7 and 24 hours after the single treatment of ONO-5390556 with the doses of 0.06 and 0.6 mg/kg. The collected tumors were disrupted and phosphorylated TrkA in tumors was detected by Electrochemiluminescence (ECL). In a PIA assay, KM12 was incubated in plasma from the collected blood for 4 hours and phosphorylated TrkA in the cells was detected by ECL.. Results: Treatment with ONO-5390556 at doses of 0.06, 0.6 mg/kg resulted in a significant inhibition of Trk phosphorylation in tumors up to 24 hours. Compared to inhibition of P-TRK in tumors, the inhibition of P-TRK in PIA sustained until 7 hours but rapidly decreased at 24 hours after administration. The levels of both tumors and PIA showed in a dose-dependent inhibition and an excellent correlation until 7 hours. Inhibition of TRK phosphorylation (PIA/Tumor, %)0.06 mg/kg (2,4,7,24h) : 56.3/41.4 (PIA/Tumor, %), 51.9/53.5, 43.0/66.5, -5.5/51.30.6 mg/kg (2,4,7,24h) : 90.2/84.3, 88.4/93.0, 87.4/95.7, 8.3/83.3Conclusion: We have validated the PIA assay that correlates with the inhibition of P-TRK in tumors. Our results demonstrated that the potential utility of PIA as a PD marker may contribute to determining the effective dose of ONO-5390556 in the clinical development. Citation Format: Ryohei Kozaki, Ryu Fujikawa, Hikaru Kato, Natsuka Goto, Toshio Yoshizawa. Plasma inhibitory activity (PIA) is a possible pharmacodynamic marker for clinical development of a next generation pan-TRK inhibitor, ONO-5390556 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2017-3727

SummaryThe 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

Breast cancer is the most common malignancy among women and has poor survival and high recurrence rates for aggressive metastatic disease. Notably, triple-negative breast cancer (TNBC) is a highly aggressive cancer and there is no preferred agent for TNBC therapy. In this study, we show that a novel agent, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109), has ability to inhibit breast cancer cell growth and invasiveness in vitro and in vivo. In addition, YL-109 repressed the sphere-forming ability and the expression of stem cell markers in MDA-MB-231 mammosphere cultures. YL-109 increased the expression of carboxyl terminus of Hsp70-interacting protein (CHIP), which suppresses tumorigenic and metastatic potential of breast cancer cells by inhibiting the oncogenic pathway. YL-109 induced CHIP transcription because of the recruitment of the aryl hydrocarbon receptor (AhR) to upstream of CHIP gene in MDA-MB-231 cells. Consistently, the antitumor effects of YL-109 were depressed by CHIP or AhR knockdown in MDA-MB-231 cells. Taken together, our findings indicate that a novel agent YL-109 inhibits cell growth and metastatic potential by inducing CHIP expression through AhR signaling and reduces cancer stem cell properties in MDA-MB-231 cells. It suggests that YL-109 is a potential candidate for breast cancer therapy.

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.