Citations

Key pointsThe gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown.In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells.Activation of Piezo2 by mechanical forces results in a characteristic ionic current, the release of serotonin and stimulation of gastrointestinal secretion.Piezo2 inhibition by drugs or molecular knockdown decreases mechanosensitive currents, serotonin release and downstream physiological effects.The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction.AbstractThe enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5‐hydroxytryptamine; 5‐HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5‐HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5‐HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically‐sensitive inward non‐selective cation current characteristic of Piezo2. Both inward currents and 5‐HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+and D‐GsMTx4). Jejunum mucosal pressure increased 5‐HT release and short‐circuit current via submucosal 5‐HT3 and 5‐HT4 receptors. Pressure‐induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D‐GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5‐HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.

Abstract Cholesterol metabolism has been implicated in prostate cancer pathogenesis. Here, we assessed the association of intratumoral mRNA expression of cholesterol synthesis enzymes, transporters, and regulators in tumor specimen at diagnosis and lethal prostate cancer, defined as mortality or metastases from prostate cancer in contrast to nonlethal disease without evidence of metastases after at least 8 years of follow-up. We analyzed the prospective prostate cancer cohorts within the Health Professionals Follow-up Study (n = 249) and the Physicians' Health Study (n = 153) as well as expectantly managed patients in the Swedish Watchful Waiting Study (n = 338). The expression of squalene monooxygenase (SQLE) was associated with lethal cancer in all three cohorts. Men with high SQLE expression (>1 standard deviation above the mean) were 8.3 times (95% confidence interval, 3.5 to 19.7) more likely to have lethal cancer despite therapy compared with men with the mean level of SQLE expression. Absolute SQLE expression was associated with lethal cancer independently from Gleason grade and stage, as was a SQLE expression ratio in tumor versus surrounding benign prostate tissue. Higher SQLE expression was tightly associated with increased histologic markers of angiogenesis. Collectively, this study establishes the prognostic value of intratumoral cholesterol synthesis as measured via SQLE, its second rate-limiting enzyme. SQLE expression at cancer diagnosis is prognostic for lethal prostate cancer both after curative-intent prostatectomy and in a watchful waiting setting, possibly by facilitating micrometastatic disease. Cancer Res; 76(16); 4785–90. ©2016 AACR.

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest malignancies lacking effective therapeutic strategies. Here we show that the non-canonical IκB-related kinase, IKBKE, acts downstream of the Gli transcription factors and engages in reciprocal regulation of Gli signaling in pancreatic cancer cells. We find that loss of IKBKE inhibits the initiation and progression of pancreatic tumors in mice carrying pancreatic specific KRAS activation. Mechanistically, IKBKE does not play a major role in NF-κB activation in pancreatic cancer; but is critical for AKT activation in vivo. Moreover, we find that IKBKE promotes reactivation of AKT post-inhibition of mTOR in PDAC cells, and that combined inhibition of IKBKE and mTOR synergistically blocks pancreatic tumor growth. Together, our findings highlight the functional interaction of IKBKE with Hh/Gli and mTOR/AKT signaling in pancreatic cancer, and suggest IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic. Citation Format: Mihir Rajurkar, Martin Fernandez-Zapico, Brian Lewis, Junhao Mao. IKBKE integrates Gli and mTOR-AKT activation during Kras-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B04.

Abstract Sphingosine kinase 1 (SphK1) phosphorylates pro-apoptotic sphingosine to form pro-survival sphingosine-1-phosphate (S1P), leading to cancer progression. In breast cancer, high levels of SphK1 mRNA are found in 80% of the patients, with average increase of 2-fold in breast tumors compared with normal tissue from the same patient. Recent studies suggest that SphK1 expression is associated with disease-free survival in ER-negative and HER2-positive breast tumors. In addition, inhibition of SphK1 in HER2-positive breast cancer cell line reduced S1P-induced HER2 and ERK1/2 activation. While these evidences suggest that SphK1 may play an important role in HER2-positive breast cancer, the exact role of SphK1 in breast tumorigenesis remains unclear. The aim of this project is to define the role of SphK1 in HER2-positive breast tumorigenesis and to investigate whether the pathway components are potential target for breast cancer prevention and therapies. Here, we report that SphK1 is required for HER2/neu-induced breast tumorigenesis and SphK1 regulates claudin-2 (CLDN2) to promote tumor development perhaps by mediating HER2. In MMTV-neu transgenic mouse model, genetic deletion of SphK1, which was accompanied by reduced S1P and increased C16-ceramide levels, significantly inhibited breast tumor development, with reduced incidence (SphK1-/-, 19%; SphK1+/-, 26%; and SphK1+/+, 57%; p < 0.05) and multiplicity (SphK1-/-, 0.27 ± 0.13; SphK1+/-, 0.32 ± 0.07; and SphK1+/+, 0.67 ± 0.14 tumors per animal; p < 0.05). Unexpectedly, SphK1 deletion did not affect tumor growth as results showed no differences in tumor volume. However, this is not surprising when considering the fact that the tumors were allowed to grow until tumor reached 20 mm in longer diameter, at which they were sacrificed regardless of age. Interestingly, whole genome expression analysis by Illumina MouseWG-6 v2.0 Expression BeadChip Array revealed significant reduction of CLDN2 expression in tumors from SphK1 deficient mice (p < 0.0001). In support to these findings, overexpression of SphK1 in MDA-MB-453 human breast cancer cells, which HER2 is amplified but lacks hormone receptors, lead to increase in CLDN2 expression (p < 0.05). Furthermore, overexpression of SphK1 in MDA-MB-453 cells also enhanced cell proliferation, colony formation, and migration/invasion, and drug resistance to cisplatin. The relevance of this finings in humans was confirmed by immunostaining of SphK1 and CLDN2 in HER2-positive breast cancer tumors and normal tissues (n = 93 and 35 subjects, respectively). The results showed that the expression of SphK1 was higher in tumors compared to adjacent normal tissue (p < 0.0001). Furthermore, higher SphK1 expression was observed in high grade tumors (vs low grade; p < 0.001) and ER-PR- tumors (vs ER+PR+; p < 0.0001). Interestingly, we observed similar staining pattern of CLDN2 proteins, where CLDN2 expression was high in tumors (p < 0.0001), particularly in high grade tumors (p < 0.01) and ER-PR- tumors (p < 0.0001). Moreover, we found a strong correlation between SphK1 and CLDN2 expressions in tumors but not in normal tissues, confirming the results obtained in animal study. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis, and targeting SphK1 and/or CLDN2 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment. Citation Format: Yoshiko Shimizu, Hideki Furuya, Paulette M. Tamashiro, Steve Goodison, Owen Chan, Ian Pagano, Kayoko Iino, Rafael Peres, Kanani Hokutan, Lenora W.M. Loo, Brenda Hernandez, Aung Naing, Charles J. Rosser, Toshihiko Kawamori. Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B17.

Abstract Purpose: Somatic mosaic mutation in the PPM1D gene have been associated with risk of breast and ovarian cancer. Somatic mosaic mutations in blood cells may be associated with older age and chemotherapy exposure. We wanted to determine if somatic mosaic mutations in PPM1D and in other genes are associated with older age and repeated exposure to chemotherapy, rather than a contributor to risk of disease. Experimental design: We used targeted capture and massively parallel genomic sequencing to sequence DNA from PBMCs from affected patients for PPM1D as well as 60 other DNA repair genes. For a number of samples with PPM1D mutations, we re-sequenced at greater depth on patients where PBMCs that were collected at diagnosis and recurrence. Results: We identified somatic mosaic mutations in exon 6 of PPM1D in 4 out of 474 (0.8%) individuals that were newly diagnosed and had no previous chemotherapy, in 3 out of 29 (10%) individuals newly diagnosed but receiving chemotherapy and in 58 of 274 (21%) individuals with relapsed platinum resistant ovarian cancer (p<0.0001). The presence of PPM1D mutations in exon 6 was strongly associated with more advanced age (p=0.011, Wilcoxon test) and more than one chemotherapy regimen (p=0.026, Fisher's exact test). In paired blood samples from 13 patients, PPM1D mutations present at relapse were either not detected or detected at a lower percentage compared to blood obtained soon after diagnosis. Somatic mosaic mutations were also identified in TP53 in 7 of 274 (2.5%) women with relapsed platinum resistant cancer. In one of these cases, we had an earlier blood sample from diagnosis in which the mutation was not present at time near diagnosis. Conclusion: We found that somatic mosaic PPM1D mutations in PMBCs are associated with advanced age and repeated exposure to chemotherapy. Mosaic mutations in other genes were not confirmed with the exception of TP53, for which mutations are also detectable after extensive treatment. It seems likely that mosaic PPM1D mutations in blood from cancer patients may be a result of selection by chemotherapy of age-induced somatic mutations during exposure to chemotherapy. Further studies are needed to determine the clinical significance of these mutations. Citation Format: Maria I. Harrell, Barbara M. Norquist, Tom Walsh, Mark F. Brady, Ming K. Lee, Robert Hershberg, Kimberly R. Kalli, Heather Lankes, Eric Q. Konnick, Colin C. Pritchard, Bradley J. Monk, John K. Chan, Robert Burger, Scott H. Kaufmann, Michael J. Birrer, Elizabeth M. Swisher. Old age and chemotherapy contribute to the selection of PPM1D somatic mosaic mutations in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B71.

Abstract Background: Previous failed trials of anti-EGFR therapy (TBCRC001) in breast cancer have not directly assessed EGFR amplification instead relying on pathway activation surrogates. Previous studies have examined EGFR amplification primarily retrospectively for research purposes without being part of a standard diagnostic evaluation. We evaluated 5,606 clincally advanced breast cases for which comprehensive genomic profiling was performed in the course of clinical care to identify EGFR altered mBC cases. Methods: DNA was extracted from 40 microns of FFPE sections from 5,605 mBC. Comprehensive genomic profiling (CGP) was performed using a hybrid-capture, adaptor ligation based next generation sequencing assay of up to 236 or 315 genes to a mean coverage depth of >600X. The results were analyzed for base substitutions, short insertions and deletions, selected rearrangements, and copy number changes. Results: 155 (2.7%) of 5,605 mBC featured EGFR alterations. 126 (10.6%) featured EGFR amplification, and 27 (2.2%) featured ERBB2 subs/indels, with no cases harboring both amplification and another GA of EGFR. These patient (pts) had a median age of 55 years (range 30 to 78). For EGFR altered cases, specimens utilized for CGP included 59 (38%) from the patient's primary BC and 93 (60%) from metastatic sites including liver (14%), lymph node (14%), lung (8%), soft tissue (6%), skin (5%) and with information not available for 3 cases. 75 (48%) mBC were submitted as IDC, 69 (45%) as breast carcinoma NOS, 5 (3 %) as metaplastic BC, and 7 (5%) as other mBC. For the 126 EGFR amplified breast patients, quantitative estimation of EGFR copy number ranged from 6 to 380 copies, with a median of 12 copies. A patient with heavily pretreated case and whose TNBC harbored 32 copies of EGFR was begun on erlotinib and has experienced a 20 months ongoing complete response to erlotinib, and will be presented as will other outcomes and biomarker characterization of these cases. Conclusions: Given anecdotal evidence that mBC is responsive to anti-EGFR targeted therapies, prediction of benefit from such therapies may be linked to sensitive and specific detection of EGFR alteration in mBC cases. This study demonstrates a 2.7% EGFR alteration rate of mBC, with a striking median copy number of 12 for the subset of EGFR amplified cases. Given these findings along with anecdotal report of patient benefit, utilization of CGP in the course of clinical care or for clinical trials for mBC may help optimize treatement with targeted therapies for these patients including the direction of anti-EGFR monotherapy to EGFR altered patients. Citation Format: Ali SM, Wang K, Johnson A, Rodriguez AA, Elvin JA, Vergilio J-A, Suh JH, Chumsri S, Morosini D, Yelensky R, Lipson D, Chmielecki J, Miller VA, Ross JS, Chang J, Stephens PJ. EGFR genomic alterations in 5,605 cases of refractory and metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-02.

Abstract Background: Abemaciclib, a small molecule inhibitor of CDK4 and CDK6, induces G1 cell cycle arrest in Rb-proficient human cancers.1 The clinical safety profile of abemaciclib enables continuous oral dosing to achieve sustained target inhibition, resulting in single-agent antitumor activity against multiple human cancers. The drug also reaches relevant concentrations in the central nervous system and, in patients taking the drug orally, can be detected in the cerebrospinal fluid.2 For women with previously treated hormone receptor positive (HR+) metastatic breast cancer (MBC), abemaciclib as a single agent achieved a six-month clinical benefit rate of 61.1% and an objective response rate of 33.3%.3 Clinical trials investigating abemaciclib combined with fulvestrant4 or aromatase inhibitors5 have led to randomized Phase 3 studies for women with HR+ breast cancer.6,7 Methods: This Phase 1b study (NCT02057133) with multiple cohorts evaluates safety and tolerability of abemaciclib combined with endocrine or HER2-targeted therapies for MBC. Secondary objectives include pharmacokinetics (PK) and antitumor activity of abemaciclib when given in combination with other therapies. Cohorts were opened to enrollment sequentially. Patients with HR+ HER2 negative MBC received abemaciclib orally every 12 hours (Q12H) in combination with the following standard therapies daily until progression: letrozole (Part A), anastrozole (Part B), tamoxifen (Part C), exemestane (Part D), or exemestane plus everolimus (Part E). Patients with HER2 positive MBC received abemaciclib orally Q12H in combination with trastuzumab every 21 days until progression (Part F). Adverse events (AEs) were graded by NCI CTCAE v4.0 and tumor response was assessed radiographically using RECIST v1.1. Results: Abemaciclib has been combined with multiple targeted therapies for the treatment of women with MBC. We previously reported safety and early efficacy results for the combinations of abemaciclib with letrozole, anastrozole, tamoxifen, exemestane, and exemestane plus everolimus.5 Due to limited follow-up at that time, the efficacy results were not mature. Safety, PK, and efficacy results with approximately 6 months of additional follow-up will be reported across all parts of the study. The most common treatment-emergent AEs include effects on the gastrointestinal and hematopoietic systems. Consistent with previously reported results for both single-agent abemaciclib and the combination of abemaciclib with fulvestrant, tumor responses have been observed among women receiving abemaciclib in combination with targeted therapies for MBC. Conclusions: This study for women with MBC demonstrates the potential for abemaciclib to be combined with therapies targeting specific signaling pathways. References: 1Gelbert et al. Invest New Drugs. 2014;32(5):825-37. 2Shapiro et al. J Clin Oncol 31, 2013 (suppl; abstr 2500). 3Tolaney et al. SABCS 2014: Abstract 763. 4Patnaik et al. J Clin Oncol 32:5s, 2014 (suppl; abstr 534). 5Tolaney et al. J Clin Oncol 33, 2015 (suppl; abstr 522). 6Llombart et al. SABCS 2014: OT1-1-07 (MONARCH 2, NCT02107703). 7Goetz et al. J Clin Oncol 33, 2015 (suppl; abstr TPS624) (MONARCH 3, NCT02246621). Citation Format: Goetz MP, Beeram M, Beck T, Conlin AK, Dees EC, Dickler MN, Helsten TL, Conkling PR, Edenfield WJ, Richards DA, Turner PK, Cai N, Chan EM, Pant S, Becerra CH, Kalinsky K, Puhalla SL, Rexer BN, Burris HA, Tolaney SM. Abemaciclib, an inhibitor of CDK4 and CDK6, combined with endocrine and HER2-targeted therapies for women with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-25.