Acetylation-dependent regulation of Skp2 function.

Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.

Light-Activated Nanodiamond-Based Drug Delivery Systems for Spatiotemporal Release of Antisense Oligonucleotides.

Nanodiamonds (NDs) are considered promising delivery platforms, but inaccurate and uncontrolled release of drugs at target sites is the biggest challenge of NDs in precision medicine. This study presents the development of phototriggerable ND-based drug delivery systems, utilizing ortho-nitrobenzyl (o-NB) molecules as photocleavable linkers between drugs and nanocarriers. UV irradiation specifically cleaved o-NB molecules and then was followed by releasing antisense oligonucleotides from ND-based carriers in both buffer and cellular environments. This ND system carried cell nonpermeable therapeutic agents for bypassing lysosomal trapping and degradation. The presence of fluorescent nitrogen-vacancy centers also allowed NDs to serve as biological probes for tracing in cells. We successfully demonstrated phototriggered release of antisense oligonucleotides from ND-based nanocarriers, reactivating their antisense functions. This highlights the potential of NDs, photocleavable linkers, and light stimuli to create advanced drug delivery systems for controlled drug release in disease therapy, opening possibilities for targeted and personalized treatments.

Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer.

High Throughput Confined Migration Microfluidic Device for Drug Screening.

Cancer metastasis is the major cause of cancer-related death. Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors, creating confined spaces for tumor cell migration and metastasis. Confined migration is involved in all metastasis steps. However, confined and unconfined migration inhibitors are different and drugs available to inhibit confined migration are rare. The main challenges are the modeling of confined migration, the suffering of low throughput, and others. Microfluidic device has the advantage to reduce reagent consumption and enhance throughput. Here, a microfluidic chip that can achieve multi-function drug screening against the collective migration of cancer cells under confined environment is designed. This device is applied to screen out effective drugs on confined migration among a novel mechanoreceptors compound library (166 compounds) in hepatocellular carcinoma, non-small lung cancer, breast cancer, and pancreatic ductal adenocarcinoma cells. Three compounds that can significantly inhibit confined migration in pan-cancer: mitochonic acid 5 (MA-5), SB-705498, and diphenyleneiodonium chloride are found. Finally, it is elucidated that these drugs targeted mitochondria, actin polymerization, and cell viability, respectively. In sum, a high-throughput microfluidic platform for screening drugs targeting confined migration is established and three novel inhibitors of confined migration in multiple cancer types are identified.

Akt-ing up on SRPK1: oncogene or tumor suppressor?

In this issue of Molecular Cell, Wang et al. (2014) report that the splicing kinase SRPK1 can function as both an oncogene and a tumor suppressor by modulating the activation state of the protein kinase Akt. This is shown to be mediated by the ability of SRPK1 to bind to the Akt phosphatase PHLPP1.

mTOR drives its own activation via SCF(ßTrCP)-dependent degradation of the mTOR inhibitor DEPTOR.

The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCF(ßTrCP). DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds ßTrCP. Failure to degrade DEPTOR through either degron mutation or ßTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.

The actin-bundling protein palladin is an Akt1-specific substrate that regulates breast cancer cell migration.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently deregulated in cancer. Downstream of PI3K, Akt1 and Akt2 have opposing roles in breast cancer invasive migration, leading to metastatic dissemination. Here, we identify palladin, an actin-associated protein, as an Akt1-specific substrate that modulates breast cancer cell invasive migration. Akt1, but not Akt2, phosphorylates palladin at Ser507 in a domain that is critical for F-actin bundling. Downregulation of palladin enhances migration and invasion of breast cancer cells and induces abnormal branching morphogenesis in 3D cultures. Palladin phosphorylation at Ser507 is required for Akt1-mediated inhibition of breast cancer cell migration and also for F-actin bundling, leading to the maintenance of an organized actin cytoskeleton. These findings identify palladin as an Akt1-specific substrate that regulates cell motility and provide a molecular mechanism that accounts for the functional distinction between Akt isoforms in breast cancer cell signaling to cell migration.

An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification.

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/µL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

Distinguishing high-metastasis-potential circulating tumor cells through fluidic shear stress in a bloodstream-like microfluidic circulatory system.

Circulating tumor cells (CTCs) play a critical role as initiators in tumor metastasis, which unlocks an irreversible process of cancer progression. Regarding the fluid environment of intravascular CTCs, a comprehensive understanding of the impact of hemodynamic shear stress on CTCs is of profound significance but remains vague. Here, we report a microfluidic circulatory system that can emulate the CTC microenvironment to research the responses of typical liver cancer cells to varying levels of fluid shear stress (FSS). We observe that HepG2 cells surviving FSS exhibit a marked overexpression of TLR4 and TPPP3, which are shown to be associated with the colony formation, migration, and anti-apoptosis abilities of HepG2. Furthermore, overexpression of these two genes in another liver cancer cell line with normally low TLR4 and TPPP3 expression, SK-Hep-1 cells, by lentivirus-mediated transfection also confirms the critical role of TLR4 and TPPP3 in improving colony formation, migration, and survival capability under a fluid environment. Interestingly, in vivo experiments show SK-Hep-1 cells, overexpressed with these genes, have enhanced metastatic potential to the liver and lungs in mouse models via tail vein injection. Mechanistically, TLR4 and TPPP3 upregulated by FSS may increase FSS-mediated cell survival and metastasis through the p53-Bax signaling pathway. Moreover, elevated levels of these genes correlate with poorer overall survival in liver cancer patients, suggesting that our findings could offer new therapeutic strategies for early cancer diagnosis and targeted treatment development.

TNA-Mediated Antisense Strategy to Knockdown Akt Genes for Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) remains a significant challenge in terms of treatment, with limited efficacy of chemotherapy due to side effects and acquired drug resistance. In this study, a threose nucleic acid (TNA)-mediated antisense approach is employed to target therapeutic Akt genes for TNBC therapy. Specifically, two new TNA strands (anti-Akt2 and anti-Akt3) are designed and synthesized that specifically target Akt2 and Akt3 mRNAs. These TNAs exhibit exceptional enzymatic resistance, high specificity, enhance binding affinity with their target RNA molecules, and improve cellular uptake efficiency compared to natural nucleic acids. In both 2D and 3D TNBC cell models, the TNAs effectively inhibit the expression of their target mRNA and protein, surpassing the effects of scrambled TNAs. Moreover, when administered to TNBC-bearing animals in combination with lipid nanoparticles, the targeted anti-Akt TNAs lead to reduced tumor sizes and decreased target protein expression compared to control groups. Silencing the corresponding Akt genes also promotes apoptotic responses in TNBC and suppresses tumor cell proliferation in vivo. This study introduces a novel approach to TNBC therapy utilizing TNA polymers as antisense materials. Compared to conventional miRNA- and siRNA-based treatments, the TNA system holds promise as a cost-effective and scalable platform for TNBC treatment, owing to its remarkable enzymatic resistance, inexpensive synthetic reagents, and simple production procedures. It is anticipated that this TNA-based polymeric system, which targets anti-apoptotic proteins involved in breast tumor development and progression, can represent a significant advancement in the clinical development of effective antisense materials for TNBC, a cancer type that lacks effective targeted therapy.

3D Biomimetic Models to Reconstitute Tumor Microenvironment In Vitro: Spheroids, Organoids, and Tumor-on-a-Chip.

Decades of efforts in engineering in vitro cancer models have advanced drug discovery and the insight into cancer biology. However, the establishment of preclinical models that enable fully recapitulating the tumor microenvironment remains challenging owing to its intrinsic complexity. Recent progress in engineering techniques has allowed the development of a new generation of in vitro preclinical models that can recreate complex in vivo tumor microenvironments and accurately predict drug responses, including spheroids, organoids, and tumor-on-a-chip. These biomimetic 3D tumor models are of particular interest as they pave the way for better understanding of cancer biology and accelerating the development of new anticancer therapeutics with reducing animal use. Here, the recent advances in developing these in vitro platforms for cancer modeling and preclinical drug screening, focusing on incorporating hydrogels are reviewed to reconstitute physiologically relevant microenvironments. The combination of spheroids/organoids with microfluidic technologies is also highlighted to better mimic in vivo tumors and discuss the challenges and future directions in the clinical translation of such models for drug screening and personalized medicine.

NFAT promotes carcinoma invasive migration through glypican-6.

Invasive migration of carcinoma cells is a prerequisite for the metastatic dissemination of solid tumours. Numerous mechanisms control the ability of cancer cells to acquire a motile and invasive phenotype, and subsequently degrade and invade the basement membrane. Several genes that are up-regulated in breast carcinoma are responsible for mediating the metastatic cascade. Recent studies have revealed that the NFAT (nuclear factor of activated T-cells) is a transcription factor that is highly expressed in aggressive breast cancer cells and tissues, and mediates invasion through transcriptional induction of pro-invasion and migration genes. In the present paper we demonstrate that NFAT promotes breast carcinoma invasion through induction of GPC (glypican) 6, a cell-surface glycoprotein. NFAT transcriptionally regulates GPC6 induction in breast cancer cells and binds to three regulatory elements in the GPC6 proximal promoter. Expression of GPC6 in response to NFAT signalling promotes invasive migration, whereas GPC6 silencing with shRNA (small-hairpin RNA) potently blocks this phenotype. The mechanism by which GPC6 promotes invasive migration involves inhibition of canonical ß-catenin and Wnt signalling, and up-regulation of non-canonical Wnt5A signalling leading to the activation of JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Thus GPC6 is a novel NFAT target gene in breast cancer cells that promotes invasive migration through Wnt5A signalling.

Upregulation of Receptor Tyrosine Kinase Activity and Stemness as Resistance Mechanisms to Akt Inhibitors in Breast Cancer.

The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial-mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer.

Cellular uptake, tissue penetration, biodistribution, and biosafety of threose nucleic acids: Assessing in vitro and in vivo delivery.

Compared with siRNAs or other antisense oligonucleotides (ASOs), the chemical simplicity, DNA/RNA binding capability, folding ability of tertiary structure, and excellent physiological stability of threose nucleic acid (TNA) motivate scientists to explore it as a novel molecular tool in biomedical applications. Although ASOs reach the target cells/tumors, insufficient tissue penetration and distribution of ASOs result in poor therapeutic efficacy. Therefore, the study of the time course of drug absorption, biodistribution, metabolism, and excretion is of significantly importance. In this work, the pharmacokinetics and biosafety of TNAs in living organisms are investigated. We found that synthetic TNAs exhibited excellent biological stability, low cytotoxicity, and substantial uptake in living cells without transfection. Using U87 three-dimensional (3D) multicellular spheroids to mimic the in vivo tumor microenvironment, TNAs showed their ability to penetrate efficiently throughout the whole multicellular spheroid as a function of incubation time and concentration when the size of the spheroid is relatively small. Additionally, TNAs could be safely administrated into Balb/c mice and most of them distributed in the kidneys where they supposed to excrete from the body through the renal filtration system. We found that accumulation of TNAs in kidneys induced no pathological changes, and no acute structural and functional damage in renal systems. The favourable biocompatibility of TNA makes it attractive as a safe and effective nucleic acid-based therapeutic agent for practical biological applications.

ANLN Enhances Triple-Negative Breast Cancer Stemness Through TWIST1 and BMP2 and Promotes its Spheroid Growth.

ANLN is frequently upregulated in triple-negative breast cancer (TNBC) and its high expression in tumors are significantly associated with poor survival and recurrence, thereby it has been proposed to function as a prognostic marker for breast cancer. However, the specific function and molecular mechanisms by which ANLN promotes TNBC tumorigenesis remain elusive. Using multiomic profiling, we recently uncovered ANLN as a TNBC-specific gene driven by super-enhancer. Here, by Crispr/Cas9 editing, we showed that knockout of ANLN inhibits spheroid growth of TNBC. Interestingly, its effect on cell proliferation in 2D cultures is minimal. ANLN depletion inhibits mammosphere formation and clonogenicity potently, suggesting its important function in regulating cancer stem cells (CSCs). We screened a panel of stem cell-related genes and uncovered several CSC genes regulated by ANLN. We further identify TWIST1 and BMP2 as essential genes that mediate ANLN's function in stemness but not spheroid growth. These findings may contribute to search for effective targeted therapies to treat TNBC.

Defining super-enhancer landscape in triple-negative breast cancer by multiomic profiling.

Breast cancer is a heterogeneous disease, affecting over 3.5 million women worldwide, yet the functional role of cis-regulatory elements including super-enhancers in different breast cancer subtypes remains poorly characterized. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis. Here we apply integrated epigenomic and transcriptomic profiling to uncover super-enhancer heterogeneity between breast cancer subtypes, and provide clinically relevant biological insights towards TNBC. Using CRISPR/Cas9-mediated gene editing, we identify genes that are specifically regulated by TNBC-specific super-enhancers, including FOXC1 and MET, thereby unveiling a mechanism for specific overexpression of the key oncogenes in TNBC. We also identify ANLN as a TNBC-specific gene regulated by super-enhancer. Our studies reveal a TNBC-specific epigenomic landscape, contributing to the dysregulated oncogene expression in breast tumorigenesis.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Akt/protein kinase b and glycogen synthase kinase-3beta signaling pathway regulates cell migration through the NFAT1 transcription factor.

The phosphoinositide 3-kinase (PI3K) pathway regulates a multitude of cellular processes. Deregulation of PI3K signaling is often observed in human cancers. A major effector of PI3K is Akt/protein kinase B (PKB). Recent studies have pointed to distinct roles of Akt/PKB isoforms in cancer cell signaling. Studies have shown that Akt1 (PKBalpha) can attenuate breast cancer cell motility, whereas Akt2 (PKBbeta) enhances this phenotype. Here, we have evaluated the mechanism by which Akt1 blocks the migration of breast cancer cells through the transcription factor NFAT. A major effector of Akt/PKB is glycogen synthase kinase-3beta (GSK-3beta), also a NFAT kinase. Inhibition of GSK-3beta using short hairpin RNA or a selective inhibitor potently blocks breast cancer cell migration concomitant with a reduction in NFAT activity. GSK-3beta-mediated inhibition of NFAT activity is due to proteasomal degradation. Experiments using GSK-3beta mutants, which are unresponsive to Akt/PKB, reveal that inhibition of cell migration by Akt/PKB is mediated by GSK-3beta. These effects are recapitulated at the levels of NFAT degradation by the proteasome. Our studies show that activation of Akt/PKB leads to inactivation of the effector GSK-3beta and the outcome of this signaling event is degradation of NFAT by the proteasome and subsequent inhibition of cell migration.

Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer.

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor-resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor-resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964-74. ©2016 AACR.