Ryoichi Matsunuma

Human Breast Cancer Tissues Contain Abundant Phosphatidylcholine(36∶1) with High Stearoyl-CoA Desaturase-1 Expression

Breast cancer is the leading cause of cancer and mortality in women worldwide. Recent studies have argued that there is a close relationship between lipid synthesis and cancer progression because some enzymes related to lipid synthesis are overexpressed in breast cancer tissues. However, lipid distribution in breast cancer tissues has not been investigated. We aimed to visualize phosphatidylcholines (PCs) and lysoPCs (LPCs) in human breast cancer tissues by performing matrix assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a novel technique that enables the visualization of molecules comprehensively. Twenty-nine breast tissue samples were obtained during surgery and subjected to MALDI-IMS analysis. We evaluated the heterogeneity of the distribution of PCs and LPCs on the tissues. Three species [PC(32∶1), PC(34∶1), and PC(36∶1)] of PCs with 1 mono-unsaturated fatty acid chain and 1 saturated fatty acid chain (MUFA-PCs) and one [PC(34∶0)] of PCs with 2 saturated fatty acid chains (SFA-PC) were relatively localized in cancerous areas rather than the rest of the sections (named reference area). In addition, the LPCs did not show any biased distribution. The relative amounts of PC(36∶1) compared to PC(36∶0) and that of PC(36∶1) to LPC(18∶0) were significantly higher in the cancerous areas. The protein expression of stearoyl-CoA desaturase-1 (SCD1), which is a synthetic enzyme of MUFA, showed accumulation in the cancerous areas as observed by the results of immunohistochemical staining. The ratios were further analyzed considering the differences in expressions of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and Ki67. The ratios of the signal intensity of PC(36∶1) to that of PC(36∶0) was higher in the lesions with positive ER expression. The contribution of SCD1 and other enzymes to the formation of the observed phospholipid composition is discussed.

UV damage-induced phosphorylation of HBO1 triggers CRL4DDB2-mediated degradation to regulate cell proliferation

ABSTRACT Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation.

Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair

HBO1, a histone acetyl transferase, is a co-activator of DNA pre-replication complex formation. We recently reported that HBO1 is phosphorylated by ATM and/or ATR and binds to DDB2 after ultraviolet irradiation. Here, we show that phosphorylated HBO1 at cyclobutane pyrimidine dimer (CPD) sites mediates histone acetylation to facilitate recruitment of XPC at the damaged DNA sites. Furthermore, HBO1 facilitates accumulation of SNF2H and ACF1, an ATP-dependent chromatin remodelling complex, to CPD sites. Depletion of HBO1 inhibited repair of CPDs and sensitized cells to ultraviolet irradiation. However, depletion of HBO1 in cells derived from xeroderma pigmentosum patient complementation groups, XPE, XPC and XPA, did not lead to additional sensitivity towards ultraviolet irradiation. Our findings suggest that HBO1 acts in concert with SNF2H–ACF1 to make the chromosome structure more accessible to canonical nucleotide excision repair factors.

Functional Annotation of ESR1 Gene Fusions in Estrogen Receptor-Positive Breast Cancer.

SUMMARY RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective.

Endocrine Therapy in Clinical Practice

Endocrine therapy (ET) is the mainstay of treatment of estrogen receptor-positive (ER+) breast cancer both in the early-stage as in the advanced disease settings. ET targets the ER pathway by blocking the body’s ability to produce estrogen or by directly modulating the ER. Since the estrogens are produced by the ovaries in premenopausal women and by some other tissues such as fat and skin in both premenopausal and postmenopausal women, ET for premenopausal women is different from for postmenopausal women. Needless to say, therapeutic options for early breast cancer are different to metastatic setting where more drugs are approved. This chapter will mainly review the clinical use of ET through all stages of breast cancer, with special considerations on recent advances in this field like ovarian function suppression in premenopausal patients with higher-risk early-stage tumors and the incorporation of targeted therapies that aim to circumvent mechanisms of endocrine resistance in metastatic ER+ breast cancer.

Author Correction: Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair

This corrects the article DOI: 10.1038/ncomms16102.

Correction: "UV damage-induced phosphorylation of HBO1 triggers CRL4DDB2-mediated degradation to regulate cell proliferation" [Molecular and Cellular Biology 36, 3, (2016) (394-406)] doi 10.1128/MCB.00809-15

Volume 36, no. 3, p. 394 – 406, 2016, https://doi.org/10.1128/MCB.00809-15. Page 396, Fig. 1C: “ -tubulin” should read “ -actin.” Page 396, column 1, lines 2 and 3: “or absence” should be deleted. Supplemental material: The following problems with the supplemental material were found. (i) In Fig. S1A, the Myc (HBO1), Chk1pS345, and -actin bands were taken from an immunoblot from an experiment that used cells treated with hydroxyurea and inserted into the figure by mistake. (ii) In Fig. S1B, the plus and minus signs for HA-Ub and MG132 for lane 2 are reversed and the -actin internal control blot was taken from a different experiment. (iii) In the Fig. S1C legend, the units for CHX should be “ M.” (iv) In the Fig. S4 legend, the units for MG132 should be “ M.” (v) In the Fig. S5 legend, the units for propidium iodide should be “ g/ml.” Revised supplemental material is posted at https://doi.org/10.1128/MCB.00809-15.

Abstract PD2-03: Recurrent functionally diverse in-frameESR1gene fusions drive endocrine resistance in breast cancer

Background. We previously reported an alternative ESR1 somatic gain-of-function chromosomal translocation event in a patient presenting with aggressive, endocrine therapy resistant estrogen receptor (ER) positive disease, producing an in-frame fusion gene consisting of N-terminal ESR1 and the C-terminus of the Hippo pathway coactivator YAP1 (ESR1-YAP1). We recently identified another ESR1 fusion through RNA sequencing (RNA-seq) in advanced stage ER+ disease from a chest wall recurrence in a male patient that was refractory to multiple lines of treatment. Two examples of fusions discovered in primary breast cancer samples include ESR1 fused in-frame to C-terminal sequences from NOP2 (ESR1-NOP2), identified in a resistant cohort from a RNA-seq screen focused on 81 primary breast cancers from aromatase inhibitor clinical trials, and a second ESR1 fusion, fused in-frame to the entire coding sequence of POLH (ESR1-POLH), that was identified from RNA-seq analysis of 728 Cancer Genome Atlas breast samples. This current study extends our previous characterization of ESR1-YAP1 by comparing functional and pharmacological properties of these three additional ESR1 gene fusion events of both early stage and advanced breast cancers. Methods. In vitro and in vivo experiments were conducted to test ESR1 fusions to induce therapeutic resistance, and metastasis. The transcriptional and binding properties of each fusion was also examined. Pharmacological inhibition with Palbociclib, a cyclin-dependent kinase 4/6 inhibitor, was utilized to assess drug sensitivity in ESR1 fusion containing breast cancer cells and in a patient derived xenograft (PDX) model expressing ESR1-YAP1 (WHIM18). Results. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth. Immunohistochemistry revealed significantly higher numbers of ER+ cells in lungs of mice xenografted with T47D cells expressing the YAP1 and PCDH11x fusions compared to YFP control, NOP2 and POLH fusions. Results from ChIP-seq and microarray studies suggest that these two fusions promote proliferation and metastasis through genomic action by binding estrogen response elements (ERE) and subsequent gene activation. We thereby define these fusions as “canonical” fusions compared to “non-canonical” NOP2 and POLH fusions, which demonstrated dramatically decreased genomic binding ability. The non-canonical fusions induced genes associated with basal-like breast cancer and promoted HER2, EGFR, and MAPK gene expression signatures in contrast to genes associated with cell cycle/proliferation induced by canonical fusions. The proliferative ability of canonical fusion-containing ER+ cells was inhibited by Palbociclib in a dose-dependent manner. In vivo WHIM18 tumors in mice fed with Palbociclib-containing chow demonstrated significantly reduced tumor volume, growth rate, and weight compared to tumors in mice on control chow. Conclusions. In-frame ERE activating canonical fusions occur in end-stage drug resistant advanced breast cancer and can be added to ESR1 point mutations as a class of recurrent somatic mutation that may cause acquired resistance. Growth induced by these fusions can be antagonized by Palbociclib and is potentially clinically helpful. Citation Format: Lei JT, Shao J, Zhang J, Iglesia M, Cao J, Chan DW, He X, Kosaka Y, Schmidt C, Matsunuma R, Haricharan S, Crowder R, Hoog J, Phommaly C, Goncalves R, Ramalho S, Lai W-C, Hampton O, Rogers A, Tobias E, Parikh P, Davies S, Ma C, Suman V, Hunt K, Watson M, Hoadley KA, Thompson A, Chen X, Perou CM, Creighton CJ, Maher C, Ellis MJ. Recurrent functionally diverse in-frame ESR1 gene fusions drive endocrine resistance in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-03.

Abstract P6-07-05: Single-cell TOF-SIMS reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells

Background Distinguishing individual cancers according to their biochemical heterogeneity have provided much useful information to clinical site. Recently, the cancer stem cell (CSC) theory has been accepted as a concept that explains the mechanism of cancer recurrence and resistance to treatment. To characterize such particular cell populations in heterogeneous tissues, we conducted combination of fluorescence activated cell sorting (FACS) and time-of-flight secondary-ion mass spectrometry (TOF-SIMS) and applied the method to analyses of breast CSCs. TOF-SIMS, which enables to visualize the composition of molecules with mass over 100 Da that were obtained in specimen, has been employed to analyze surface of industrial materials and biomaterials. This method is thus suitable for performing single cell analysis of membranous lipids. Methods Breast cancer specimens surgically resected from two patients were enzymatically dispersed into cells. They were labeled with fluorescence-conjugated antibodies of CD45, CD44, and CD24. The cells of CD45-CD44+ CD24- were sorted as CSCs with FACS as well as CD45-CD44- CD24+ cells as non-stem cancer cells (NSCCs). TOF-SIMS analysis and fatty acid analysis was performed according to our previous study published in Surface and Interface Analysis (1). The surface of the sorted cells was analyzed by a PHI TRIFT V (ULVAC-PHI Inc., Kanagawa, Japan) TOF-SIMS instrument. Primary ion beam is irradiated to the surface of the samples and, secondary ions derived from samples are calculated by time-of-flight with the information of the place where the molecular ions were ejected. Negative secondary ions were obtained with a mass range of m/z 0–1850. Mass spectra were analyzed by WinCadenceN software (ULVAC-PHI Inc.) to obtain ion counts and ion images. Integrated ion intensities of FA were normalized using phosphoric acid intensity. The Welch’s t-test was used to compare the normalized ion counts with P-value Results FACS analyses successfully collected CD45-/CD44+/CD24- CSCs and CD45-/CD44-/CD24+ non-stem cancer cells (NSCCs) in both two cases, which were corresponding to o.33% and 0.74% of all cells in case 1, and 0.14% and 1.14% in case 2. TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CSCs and NSCCs. These ions probably came from membranous phosphopolipids and they were uniformly detected from the locus where the cell attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] of CSCs normalized by phosphoric acids signals were significantly decreased than that of CD45-/CD44-/CD24+ NSCCs as a counterpart. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified with complicated combination of marker expressions in clinical specimens composed of heterogeneous cellular populations, and characterized lipid composition of CSCs. Reference 1. Nagata Y, Ishizaki I, Waki M, Ide Y, Hossen A, Ohnishi K, et al. Glutaraldehyde Fixation Method for Single-Cell Lipid Analysis by Time-of-Flight Secondary Ion-Mass Spectrometry. Surface and interface analysis : SIA. 2014:DOI: 10.1002/sia/5522. Citation Format: Yoshimi Ide, Michihiko Waki, Itsuko Ishizaki, Yasuyuki Nagata, Yumiko Taki, Yuko Hosokawa, Ryoichi Matsunuma, Hiroyuki Ogura, Norihiko Shiiya, Noriaki Sanada, Mitsutoshi Setou. Single-cell TOF-SIMS reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-05.

P4-05-05: Imaging Mass Spectrometry Based Lipid Metabolites Analysis for Breast Cancer.

Background: Activation of lipid metabolism is an early event of carcinogenesis and a central hallmark of many cancers including breast cancer. Recent findings argue that stearoyl CoA desaturase-1 (SCD1), a key regulator of the fatty acid (FA) composition and the endoplasmic reticulum resident enzyme that converts saturated FA (SFA) into monounsaturated FA (MUFA) is a novel regulator of carcinogenesis. The distinctive lipids composition of membrane in cancer cells and the biological functions of SCD1, however, still remain uncertain. Imaging mass spectrometry (IMS) is a mass spectrometry-based analyzing technique that enables visualization of the individual molecules without requiring antibodies. It allows comprehensive detection of a wide range of biomolecules, such as lipids. We attempted to visualize the localization of lipids in breast cancer by IMS for better understanding of cancer proliferation. Materials and methods: 13 specimens were obtained from the primary breast cancer patients. All were Japanese woman and aged 41–86 years (mean 61.5y.o.). Only one patient received preoperative systematic therapy. 6 were estrogen receptor (ER) and/or progesterone receptor (PgR) positive and human epidermal growth factor receptor 2 (HER2) negative, 2 were ER and/or PgR positive and HER2 positive, 2 were both ER and PgR negative and HER2 positive and 2 were triple negative. IMS: Samples were immediately chilled in liquid Hexan and stored at −80°. All specimens were sliced into 10 mm thin sections, mounted onto one indium-tin oxide-coated glass slides (Bruker Daltonics) and then sprayed by 2,5-Dihydroxybenzoic acid. Matrix assisted laser desorption ionization (MALDI) technique was used as a soft ionization method. We used time of flight (TOF)/TOF type instrument (Ultraflex, Bruker Daltonics) and all the spectrum were acquired automatically using Fleximaging software (Bruker Daltonics). Each spectral intensity at any mass-to-charge ratio (m/z) was measured at 16 regions of interest (ROI); 13 ROI were picked up from cancerous parts and 3 were from non-cancerous parts. Spectral intensities were compared and statistical analysis was performed by Mann Whitney test. The software was also used to create two-dimensional ion-density maps. Results: In the cancerous parts of all the 13 specimens, two distinct peaks of the molecular ions were detected at m/z 798.5 and 810.5, which were not found in the non-cancerous parts. Median intensity of the molecular ions at m/z 798.5 and 810.5 were 38.9 and 3.18 in the cancerous part, while they were 0.84 and 1.02 in the non-cancerous part (p=0.010 and 0.015, respectively). Tandem mass spectrometry analysis for these two molecules revealed that they were two kinds of phosphatidylcholine (PC), PC (16:0/18:1) and PC (18:0/18:1). Localization of the individual PC was visualized by means of IMS, which showed that in cancerous part accumulation of PCs containing MUFA was more pronounced than those containing SFA only. Conclusion: Two kinds of PC containing MUFA were found to highly accumulate in cancerous parts, which may suggest involvement of SCD1 in the membrane composition regulation and cancer proliferation. Further studies may thus be warranted to explore the relation between PC localization and the SCD1 expression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-05.