Advanced breast cancer is frequently associated with skeletal metastases and accelerated bone loss. Recombinant parathyroid hormone [teriparatide, PTH(1-34)] is the first anabolic agent approved in the US for treatment of osteoporosis. While signaling through the PTH receptor in the osteoblast lineage regulates bone marrow hematopoietic niches, the effects of anabolic PTH on the skeletal metastatic niche are unknown. Here, we demonstrate, using orthotopic and intratibial models of 4T1 murine and MDA-MB-231 human breast cancer tumors, that anabolic PTH decreases both tumor engraftment and the incidence of spontaneous skeletal metastasis in mice. Microcomputed tomography and histomorphometric analyses revealed that PTH increases bone volume and reduces tumor engraftment and volume. Transwell migration assays with murine and human breast cancer cells revealed that PTH alters the gene expression profile of the metastatic niche, in particular VCAM-1, to inhibit recruitment of cancer cells. While PTH did not affect growth or migration of the primary tumor, it elicited several changes in the tumor gene expression profile resulting in a less metastatic phenotype. In conclusion, PTH treatment in mice alters the bone microenvironment, resulting in decreased cancer cell engraftment, reduced incidence of metastases, preservation of bone microarchitecture and prolonged survival.
Srilatha Swami, Joshua Johnson, Lance A. Bettinson, Takaharu Kimura, Hui Zhu, Megan A. Albertelli, Rachelle W. Johnson, Joy Y. Wu
Bone metastases (BoM) are a significant cause of morbidity in patients with estrogen receptor–positive (ER-positive) breast cancer; yet, characterizations of human specimens are limited. In this study, exome-capture RNA sequencing (ecRNA-seq) on aged (8–12 years), formalin-fixed, paraffin-embedded (FFPE), and decalcified cancer specimens was evaluated. Gene expression values and ecRNA-seq quality metrics from FFPE or decalcified tumor RNA showed minimal differences when compared with matched flash-frozen or nondecalcified tumors. ecRNA-seq was then applied on a longitudinal collection of 11 primary breast cancers and patient-matched synchronous or recurrent BoMs. Overtime, BoMs exhibited gene expression shifts to more Her2 and LumB PAM50 subtype profiles, temporally influenced expression evolution, recurrently dysregulated prognostic gene sets, and longitudinal expression alterations of clinically actionable genes, particularly in the CDK/Rb/E2F and FGFR signaling pathways. Taken together, this study demonstrates the use of ecRNA-seq on decade-old and decalcified specimens and defines recurrent longitudinal transcriptional remodeling events in estrogen-deprived breast cancers.
Nolan Priedigkeit, Rebecca J. Watters, Peter C. Lucas, Ahmed Basudan, Rohit Bhargava, William Horne, Jay K. Kolls, Zhou Fang, Margaret Q. Rosenzweig, Adam M. Brufsky, Kurt R. Weiss, Steffi Oesterreich, Adrian V. Lee
The oncoprotein Mdm2 is a RING domain–containing E3 ubiquitin ligase that ubiquitinates G protein–coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53–KO mice, which survive for 9–12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53–KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53–KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53–KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53–KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53–KO was impaired. Mdm2/p53–KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53–KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53–KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.
Pierre-Yves Jean-Charles, Samuel Mon-Wei Yu, Dennis Abraham, Reddy Peera Kommaddi, Lan Mao, Ryan T. Strachan, Zhu-Shan Zhang, Dawn E. Bowles, Leigh Brian, Jonathan A. Stiber, Stephen N. Jones, Walter J. Koch, Howard A. Rockman, Sudha K. Shenoy
MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16–/– mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16–/– mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16–/– mice failed to rescue the Mtg16–/– protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.
Elizabeth M. McDonough, Caitlyn W. Barrett, Bobak Parang, Mukul K. Mittal, J. Joshua Smith, Amber M. Bradley, Yash A. Choksi, Lori A. Coburn, Sarah P. Short, Joshua J. Thompson, Baolin Zhang, Shenika V. Poindexter, Melissa A. Fischer, Xi Chen, Jiang Li, Frank L. Revetta, Rishi Naik, M. Kay Washington, Michael J. Rosen, Scott W. Hiebert, Keith T. Wilson, Christopher S. Williams
Adrenergic signaling is known to promote tumor growth and metastasis, but the effects on tumor stroma are not well understood. An unbiased bioinformatics approach analyzing tumor samples from patients with known biobehavioral profiles identified a prominent stromal signature associated with cancer-associated fibroblasts (CAFs) in those with a high biobehavioral risk profile (high Center for Epidemiologic Studies Depression Scale [CES-D] score and low social support). In several models of epithelial ovarian cancer, daily restraint stress resulted in significantly increased CAF activation and was abrogated by a nonspecific β-blocker. Adrenergic signaling–induced CAFs had significantly higher levels of collagen and extracellular matrix components than control tumors. Using a systems-based approach, we found INHBA production by cancer cells to induce CAFs. Ablating inhibin β A decreased CAF phenotype both in vitro and in vivo. In preclinical models of breast and colon cancers, there were increased CAFs and collagens following daily restraint stress. In an independent data set of renal cell carcinoma patients, there was an association between high depression (CES-D) scores and elevated expression of ACTA2, collagens, and inhibin β A. Collectively, our findings implicate adrenergic influences on tumor stroma as important drivers of CAFs and establish inhibin β A as an important regulator of the CAF phenotype in ovarian cancer.
Archana S. Nagaraja, Robert L. Dood, Guillermo Armaiz-Pena, Yu Kang, Sherry Y. Wu, Julie K. Allen, Nicholas B. Jennings, Lingegowda S. Mangala, Sunila Pradeep, Yasmin Lyons, Monika Haemmerle, Kshipra M. Gharpure, Nouara C. Sadaoui, Cristian Rodriguez-Aguayo, Cristina Ivan, Ying Wang, Keith Baggerly, Prahlad Ram, Gabriel Lopez-Berestein, Jinsong Liu, Samuel C. Mok, Lorenzo Cohen, Susan K. Lutgendorf, Steve W. Cole, Anil K. Sood
The molecular bases for sex differences in cancer remain undefined and how to incorporate them into risk stratification remains undetermined. Given sex differences in metabolism and the inverse correlation between fluorodeoxyglucose (FDG) uptake and survival, we hypothesized that glycolytic phenotyping would improve glioma subtyping. Using retrospectively acquired lower-grade glioma (LGG) transcriptome data from The Cancer Genome Atlas (TCGA), we discovered male-specific decreased survival resulting from glycolytic gene overexpression. Patients within this high-glycolytic group showed significant differences in the presence of key genomic alterations (i.e., 1p/19q codeletion, CIC, EGFR, NF1, PTEN, FUBP1, and IDH mutations) compared with the low-glycolytic group. Although glycolytic stratification defined poor prognostic males independent of grade, histology, TP53, and ATRX mutation status, we unexpectedly found that females with high-glycolytic gene expression and wild-type IDH survived longer than all other wild-type patients. Validation with an independent metabolomics dataset from grade 2 gliomas determined that glycolytic metabolites selectively stratified males and also uncovered a potential sexual dimorphism in pyruvate metabolism. These findings identify a potential synergy between patient sex, tumor metabolism, and genomic alterations in determining outcome for glioma patients.
Joseph E. Ippolito, Aldrin Kay-Yuen Yim, Jingqin Luo, Prakash Chinnaiyan, Joshua B. Rubin
The tumor suppressor PTEN is frequently inactivated in breast and other cancers; yet, germ-line mutations in this gene induce nonmalignant hamartomas, indicating dependency on additional cooperating events. Here we show that most tumors derived from conditional deletion of mouse pten in mammary epithelium are highly differentiated and lack transplantable tumor-initiating cells (TICs) capable of seeding new tumors following orthotopic injection of FACS-sorted or tumorsphere cells. A rare group of poorly differentiated tumors did harbor transplantable TICs. These transplantable tumors exhibited distinct molecular classification, signaling pathways, chromosomal aberrations, and mutational landscape, as well as reduced expression of microRNA-143/145 (miR-143/145). Stable knockdown of miR-143/145 conferred tumorigenic potential upon poorly transplantable pten-deficient tumor cells through a mechanism involving induction of RAS signaling, leading to increased sensitivity to MEK inhibition. In humans, miR-145 deficiency significantly correlated with elevated RAS-pathway activity in basal-like breast cancer, and patients with combined PTEN/miR-145 loss or PTEN-loss/high RAS-pathway activity exhibited poor clinical outcome. These results underscore a selective pressure for combined PTEN loss together with RAS-pathway activation, either through miR-145 loss or other mechanisms, in basal-like breast cancer, and a need to identify and prioritize these tumors for aggressive therapy.
Sharon Wang, Jeff C. Liu, YoungJun Ju, Giovanna Pellecchia, Veronique Voisin, Dong-Yu Wang, Rajwinder Leha l, Yaacov Ben-David, Gary D. Bader, Eldad Zacksenhaus
BACKGROUND. Dysregulated lipid and glucose metabolism in clear cell renal cell carcinoma (ccRCC) has been implicated in disease progression, and whole tumor tissue–based assessment of these changes is challenged by the tumor heterogeneity. We studied a noninvasive quantitative MRI method that predicts metabolic alterations in the whole tumor. METHODS. We applied Dixon-based MRI for in vivo quantification of lipid accumulation (fat fraction [FF]) in targeted regions of interest of 45 primary ccRCCs and correlated these MRI measures to mass spectrometry–based lipidomics and metabolomics of anatomically colocalized tissue samples isolated from the same tumor after surgery. RESULTS. In vivo tumor FF showed statistically significant (P < 0.0001) positive correlation with histologic fat content (Spearman correlation coefficient, ρ = 0.79), spectrometric triglycerides (ρ = 0.56) and cholesterol (ρ = 0.47); it showed negative correlation with free fatty acids (ρ = –0.44) and phospholipids (ρ = –0.65). We observed both inter- and intratumoral heterogeneity in lipid accumulation within the same tumor grade, whereas most aggressive tumors (International Society of Urological Pathology [ISUP] grade 4) exhibited reduced lipid accumulation. Cellular metabolites in tumors were altered compared with adjacent renal parenchyma. CONCLUSION. Our results support the use of noninvasive quantitative Dixon-based MRI as a biomarker of reprogrammed lipid metabolism in ccRCC, which may serve as a predictor of tumor aggressiveness before surgical intervention. FUNDING. NIH R01CA154475 (YZ, MF, PK, IP), NIH P50CA196516 (IP, JB, RJD, JAC, PK), Welch Foundation I-1832 (JY), and NIH P01HL020948 (JGM).
Yue Zhang, Durga Udayakumar, Ling Cai, Zeping Hu, Payal Kapur, Eun-Young Kho, Andrea Pavía-Jiménez, Michael Fulkerson, Alberto Diaz de Leon, Qing Yuan, Ivan E. Dimitrov, Takeshi Yokoo, Jin Ye, Matthew A. Mitsche, Hyeonwoo Kim, Jeffrey G. McDonald, Yin Xi, Ananth J. Madhuranthakam, Durgesh K. Dwivedi, Robert E. Lenkinski, Jeffrey A. Cadeddu, Vitaly Margulis, James Brugarolas, Ralph J. DeBerardinis, Ivan Pedrosa
BACKGROUND. Programmed death 1 (PD-1) inhibition activates partially exhausted cytotoxic T lymphocytes (peCTLs) and induces tumor regression. We previously showed that the peCTL fraction predicts response to anti–PD-1 monotherapy. Here, we sought to correlate peCTL and regulatory T lymphocyte (Treg) levels with response to combination immunotherapy, and with demographic/disease characteristics, in metastatic melanoma patients. METHODS. Pretreatment melanoma samples underwent multiparameter flow cytometric analysis. Patients were treated with anti–PD-1 monotherapy or combination therapy, and responses determined by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) criteria. peCTL and Treg levels across demographic/disease variables were compared. Low versus high peCTL (≤20% vs. >20%) were defined from a previous study. RESULTS. One hundred and two melanoma patients were identified. The peCTL fraction was higher in responders than nonresponders. Low peCTL correlated with female sex and liver metastasis, but not with lactate dehydrogenase (LDH), tumor stage, or age. While overall response rates (ORRs) to anti–PD-1 monotherapy and combination therapy were similar in high-peCTL patients, low-peCTL patients given combination therapy demonstrated higher ORRs than those who received monotherapy. Treg levels were not associated with these factors nor with response. CONCLUSION. In melanoma, pretreatment peCTL fraction is reduced in women and in patients with liver metastasis. In low-peCTL patients, anti–PD-1 combination therapy is associated with significantly higher ORR than anti–PD-1 monotherapy. Fewer tumor-infiltrating peCTLs may be required to achieve response to combination immunotherapy. TRIAL REGISTRATION. UCSF IRB Protocol 138510 FUNDING. NIH DP2-AR068130, K08-AR062064, AR066821, and Burroughs Wellcome CAMS-1010934 (M.D.R.). Amoroso and Cook Fund, and the Parker Institute for Cancer Immunotherapy (A.I.D.).
Kimberly Loo, Katy K. Tsai, Kelly Mahuron, Jacqueline Liu, Mariela L. Pauli, Priscila M. Sandoval, Adi Nosrati, James Lee, Lawrence Chen, Jimmy Hwang, Lauren S. Levine, Matthew F. Krummel, Alain P. Algazi, Michael D Alvarado, Michael D. Rosenblum, Adil I. Daud
Evaluation of T lymphocyte frequency provides prognostic information for patients with oral squamous cell cancer (OSCC). However, the effect of simultaneously evaluating T cell frequency and assessing suppressive elements and defects in antigen-processing machinery (APM) has not been clarified. Simultaneous characterization of CD3+, CD8+, FoxP3+, CD163+, and PD-L1+ cells using multispectral imaging was performed on sections from 119 patients with HPV– OSCC. Expression of β2-microglobulin, MHC class I heavy chain, and large multifunctional peptidase 10 was quantified, and all data were correlated with patient outcome. We found that, consistent with previous reports, high numbers of CD8+ T cells at the invasive margin correlated significantly with prolonged overall survival (OS), while the number of FoxP3+ or PD-L1+ cells did not. Compiling the number of FoxP3+ or PD-L1+ cells within 30 μm of CD8+ T cells identified a significant association with a high number of suppressive elements close to CD8+ T cells and reduced OS. Integrating this information into a cumulative suppression index (CSI) increased correlation with OS. Incorporating tumor expression levels of APM components with CSI further improved prognostic power. This multiparametric immune profiling may be useful for stratifying patients with OSCC for clinical trials.
Zipei Feng, Daniel Bethmann, Matthias Kappler, Carmen Ballesteros-Merino, Alexander Eckert, R. Bryan Bell, Allen Cheng, Tuan Bui, Rom Leidner, Walter J. Urba, Kent Johnson, Clifford Hoyt, Carlo B. Bifulco, Juergen Bukur, Claudia Wickenhauser, Barbara Seliger, Bernard A. Fox
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