PI3K/mTOR inhibition can impair tumor invasion and metastasis in vivo despite a lack of antiproliferative action in vitro: implications for targeted therapy

Oncogenic PI3K/mTOR activation is frequently observed in human cancers and activates cell motility via p27 phosphorylations at T157 and T198. Here we explored the potential for a novel PI3K/mTOR inhibitor to inhibit tumor invasion and metastasis. An MDA-MB-231 breast cancer line variant, MDA-MB-231-1833, with high metastatic bone tropism, was treated with a novel catalytic PI3K/mTOR inhibitor, PF-04691502, at nM doses that did not impair proliferation. Effects on tumor cell motility, invasion, p27 phosphorylation, localization, and bone metastatic outgrowth were assayed. MDA-MB-231-1833 showed increased PI3K/mTOR activation, high levels of cytoplasmic p27pT157pT198 and increased cell motility and invasion in vitro versus parental. PF-04691502 treatment, at a dose that did not affect proliferation, reduced total and cytoplasmic p27, decreased p27pT157pT198 and restored cell motility and invasion to levels seen in MDA-MB-231. p27 knockdown in MDA-MB-231-1833 phenocopied PI3K/mTOR inhibition, whilst overexpression of the phosphomimetic mutant p27T157DT198D caused resistance to the anti-invasive effects of PF-04691502. Pre-treatment of MDA-MB-231-1833 with PF-04691502 significantly impaired metastatic tumor formation in vivo, despite lack of antiproliferative effects in culture and little effect on primary orthotopic tumor growth. A further link between cytoplasmic p27 and metastasis was provided by a study of primary human breast cancers which showed cytoplasmic p27 is associated with increased lymph nodal metastasis and reduced survival. Novel PI3K/mTOR inhibitors may oppose tumor metastasis independent of their growth inhibitory effects, providing a rationale for clinical investigation of PI3K/mTOR inhibitors in settings to prevent micrometastasis. In primary human breast cancers, cytoplasmic p27 is associated with worse outcomes and increased nodal metastasis, and may prove useful as a marker of both PI3K/mTOR activation and PI3K/mTOR inhibitor efficacy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-012-2389-6) contains supplementary material, which is available to authorized users

Expansion and Characterization of Human Melanoma Tumor-Infiltrating Lymphocytes (TILs)

Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Tri-Compartmental Restriction Spectrum Imaging Breast Model Distinguishes Malignant Lesions from Benign Lesions and Healthy Tissue on Diffusion-Weighted Imaging

Diffusion-weighted MRI (DW-MRI) offers a potential adjunct to dynamic contrast-enhanced MRI to discriminate benign from malignant breast lesions by yielding quantitative information about tissue microstructure. Multi-component modeling of the DW-MRI signal over an extended b-value range (up to 3000 s/mm2) theoretically isolates the slowly diffusing (restricted) water component in tissues. Previously, a three-component restriction spectrum imaging (RSI) model demonstrated the ability to distinguish malignant lesions from healthy breast tissue. We further evaluated the utility of this three-component model to differentiate malignant from benign lesions and healthy tissue in 12 patients with known malignancy and synchronous pathology-proven benign lesions. The signal contributions from three distinct diffusion compartments were measured to generate parametric maps corresponding to diffusivity on a voxel-wise basis. The three-component model discriminated malignant from benign and healthy tissue, particularly using the restricted diffusion C1 compartment and product of the restricted and intermediate diffusion compartments (C1 and C2). However, benign lesions and healthy tissue did not significantly differ in diffusion characteristics. Quantitative discrimination of these three tissue types (malignant, benign, and healthy) in non-pre-defined lesions may enhance the clinical utility of DW-MRI in reducing excessive biopsies and aiding in surveillance and surgical evaluation without repeated exposure to gadolinium contrast

Machine Learning and Deep Neural Networks in Thoracic and Cardiovascular Imaging.

Advances in technology have always had the potential and opportunity to shape the practice of medicine, and in no medical specialty has technology been more rapidly embraced and adopted than radiology. Machine learning and deep neural networks promise to transform the practice of medicine, and, in particular, the practice of diagnostic radiology. These technologies are evolving at a rapid pace due to innovations in computational hardware and novel neural network architectures. Several cutting-edge postprocessing analysis applications are actively being developed in the fields of thoracic and cardiovascular imaging, including applications for lesion detection and characterization, lung parenchymal characterization, coronary artery assessment, cardiac volumetry and function, and anatomic localization. Cardiothoracic and cardiovascular imaging lies at the technological forefront of radiology due to a confluence of technical advances. Enhanced equipment has enabled computed tomography and magnetic resonance imaging scanners that can safely capture images that freeze the motion of the heart to exquisitely delineate fine anatomic structures. Computing hardware developments have enabled an explosion in computational capabilities and in data storage. Progress in software and fluid mechanical models is enabling complex 3D and 4D reconstructions to not only visualize and assess the dynamic motion of the heart, but also quantify its blood flow and hemodynamics. And now, innovations in machine learning, particularly in the form of deep neural networks, are enabling us to leverage the increasingly massive data repositories that are prevalent in the field. Here, we discuss developments in machine learning techniques and deep neural networks to highlight their likely role in future radiologic practice, both in and outside of image interpretation and analysis. We discuss the concepts of validation, generalizability, and clinical utility, as they pertain to this and other new technologies, and we reflect upon the opportunities and challenges of bringing these into daily use

Abstract 1145: Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation

Abstract p27 is a cell cycle regulator that acts largely to restrain normal cell growth. It is rarely mutated or deleted in human cancers, but is often degraded or mislocalized to the cytoplasm in aggressive tumors. Phosphorylation at T157 or T198 by different PI3K effector kinases leads to p27 cytoplasmic accumulation and enhanced cell motility and invasion in part via effects on the actin cytoskeleton, which are independent of its cell cycle role. However, the functional contributions of C-terminally phosphorylated, cytoplasmic p27 to cancer progression remain poorly understood. Targeted inhibition of PI3K/mTOR impaired tumor cell motility and metastasis via modulation of p27 in a bone metastatic model (Wander, S. et al. BCRT 2013). We observed that p27 knockdown in highly metastatic cancer lines with high levels of cytoplasmic p27pT157pT198 reverted EMT and impaired tumor cell invasion in vitro and metastasis in vivo. This led us to investigate whether PI3K-activated, phosphorylated p27 may function as a novel EMT regulator. A cell cycle defective (CK-) and double phosphomimetic p27 mutant (T157D/T198D or DD) was introduced into immortal human mammary epithelial cells with low PI3K activity. This revealed a novel, oncogenic function of p27 in regulating tumor progression, in that p27CK-DD induced epithelial-mesenchymal transition (EMT) and soft agar colony formation. p27CK-DD also increased motility, invasion and formation of metastasis in bladder and breast cancer models. A RT-PCR screen for potential p27-induced EMT mediators revealed a 20-fold increase in expression of the Twist1 transcription factor in p27CK-DD-transduced cells. Knockdown of Twist reversed the p27CK-DD-mediated increase in EMT marker expression. p27 knockdown rapidly attenuated Twist1-promoter activity and reduced Twist1 mRNA levels and also increased E-cadherin expression in metastatic cells, suggesting that Twist1 may play a critical role in p27CK-DD-induced EMT. These data extend our understanding of p27 function in human cancer and suggest that PI3K deregulated p27 may promote EMT and tumor metastasis through transcriptional activation of TWIST1. Citation Format: Dekuang Zhao, Alexandra H. Besser, Wen Zhou, Seth A. Wander, Jun Sun, Michael Durante, Feng Hong, Bin Wang, Tan Ince, Karoline Briegel, Joyce M. Slingerland. Cytoplasmic p27 promotes epithelial-mesenchymal transition and tumor progression via Twist1 upregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1145. doi:10.1158/1538-7445.AM2014-1145</jats:p

Erratum to: PI3K/mTOR inhibition can impair tumor invasion and metastasis in vivo despite a lack of antiproliferative action in vitro: implications for targeted therapy

Erratum to: Breast Cancer Res Treat (2013),138:369–381,DOI 10.1007/s10549-012-2389-6. In the original publication of the article, the Fig. 4c and d were published erroneously. The revised Fig. 4 is given in this erratum

Abstract 2224: Targeted PI3K/mTOR inhibition impairs tumor cell motility and bone metastatic outgrowth via modulation of p27

Abstract The pleiotropic oncogenic effects of PI3K/mTOR have stimulated development of catalytic PI3K and mTOR inhibitors for the treatment of cancer. Present data provide a novel rationale for the use of dual PI3K/mTOR inhibitors to oppose tumor motility, invasion and metastasis. Cytoplasmic mislocalization of p27 is implicated as a key downstream driver of PI3K/mTOR-dependent tumor cell motility in vitro and bone metastatic outgrowth in vivo. Here, we report that PI3K/mTOR-kinase inhibition with PF-04691502 impairs breast cancer metastasis to bone, in part via effects on p27. Low-dose PF-04691502 impaired motility and invasion of highly PI3K/mTOR activated, bone-metastatic MDA-MB-1833 cells in vitro without affecting apoptosis or proliferation. Moreover, drug pre-treatment impaired bone metastasis in vivo. PF-04691502 decreased cytoplasmic p27pT157 and p27pT198, and p27T157D/T198D overexpression conferred resistance to inhibition of motility by PF-04691502. p27 knockdown in MDA-MB-1833 relieved RhoA-ROCK inhibition, decreased cell motility/invasion in vitro, and bone metastasis in vivo. p27 knockdown also reversed EMT phenotypic markers in MDA-MB-1833, as did PI3K/mTOR inhibition, demonstrating that p27 may be critical for maintenance of the EMT cellular program. Indeed, loss of cytoplasmic p27 reversed expression of an EMT gene profile supporting the notion that p27 acts as a key mediator of metastasis downstream of PI3K/mTOR. Cytoplasmic p27 in human cancers may prove to be a useful predictive marker to assess both PI3K/mTOR activation and the potential efficacy of catalytic PI3K/mTOR inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2224. doi:1538-7445.AM2012-2224</jats:p

MAPK Activation Predicts Poor Outcome and the MEK Inhibitor, Selumetinib, Reverses Antiestrogen Resistance in ER-Positive High-Grade Serous Ovarian Cancer

OBJECTIVE: While 67% of high grade serous ovarian cancers (HGSOC) express the estrogen receptor (ER), most fail antiestrogen therapy. Since mitogen-activated protein kinases (MAPK) activation is frequent in ovarian cancer, we investigated if estrogen regulates MAPK and if MEK inhibition (MEKi) reverses anti-estrogen resistance. METHODS: Effects of MEKi (selumetinib), anti-estrogen (fulvestrant), or both were assayed in ER+ HGSOC in vitro and in xenografts. Response biomarkers were investigated by gene expression microarray and reverse phase protein array (RPPA). Genes differentially expressed in two independent primary HGSOCs datasets with high vs low pMAPK by RPPA were used to generate a “MAPK-activated gene signature”. Gene signature components reversed by MEKi were then identified. RESULTS: High intratumor pMAPK independently predicts decreased survival (HR = 1.7, CI>95% 1.3–2.2, p=0.0009) in 408 TCGA HGSOC. A differentially expressed “MAPK-activated” gene subset was also prognostic. “MAPK-activated genes” in HGSOC differ from those in breast cancer. Combined MEK and ER blockade showed greater anti-tumor effects in xenografts than monotherapy. Gene set enrichment analysis and RPPA showed dual therapy downregulated DNA replication and cell cycle drivers, and upregulated lysosomal gene sets. Selumetinib reversed expression of a subset of “MAPK-activated genes” in vitro and/or in xenografts. Three of these genes were prognostic for poor survival (p=0.000265) and warrant testing as a signature predictive of MEKi response. CONCLUSION: High pMAPK is independently prognostic and may underlie antiestrogen failure. Data support further evaluation of fulvestrant and selumetinib in ER+ HGSOC. The MAPK-activated HGSOC signature may help identify MEK inhibitor responsive tumors