Comment on Cancer Res. 2020 Dec 1;80(23):5393-5407.

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi-independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer-related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer. SIGNIFICANCE: PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

Polyploid giant cancer cells (PGCC) are common in tumors and have been associated with resistance to cancer therapy, tumor relapse, malignancy, immunosuppression, metastasis, cancer stem cell production, and modulation of the tumor microenvironment. However, the molecular mechanisms that cause these cells to form are not yet known. In this study, we discover that Aurora kinases are synergistic determinants of a switch from the proliferative cell cycle to polyploid growth and multinucleation in lung cancer cell lines. When Aurora kinases were inhibited together, lung cancer cells uniformly grew into multinucleated PGCCs. These cells adopted an endoreplication in which the genome replicates, mitosis is omitted, and cells grow in size. Consequently, such cells continued to safely grow in the presence of antimitotic agents. These PGCC re-entered the proliferative cell cycle and grew in cell number when treatment was terminated. Thus, PGCC formation might represent a fundamental cellular response to Aurora kinase inhibitors and contributes to therapy resistance or tumor relapse. SIGNIFICANCE: These findings provide a novel insight about how cancer cells respond to Aurora kinase inhibitors and identify a new mechanism responsible for resistance to these agents and other antimitotic drugs.

MYC is a highly validated oncogenic transcription factor and cancer target. However, the disordered nature of this protein has made it a challenging target, with no clinical stage, direct small-molecule MYC inhibitors available. Recent work leveraging a large in silico chemical library and a rapid in vivo screen has expanded the chemotypes of direct small-molecule inhibitors (MYCi). Novel MYCi represent a class of improved MYC chemical probes that bind directly to MYC to inhibit its function and to promote its degradation by enhancing GSK3β-mediated phosphorylation. One of these compounds, MYCi975, has shown remarkable tolerability and efficacy in vivo and is associated with a selective effect on MYC target gene expression. Additional effects of MYCi on the tumor immune microenvironment including immune cell infiltration and upregulation of PD-L1 expression provide a rationale for combining MYCi with anti-PD-1/PD-L1 therapy to enhance antitumor efficacy. Our strategy for developing MYCi demonstrates an efficient way to identify selective and well-tolerated MYC inhibitors. The new MYCi provide tools for probing MYC function and serve as starting points for the development of novel anti-MYC therapeutics.

DZIP3/hRUL138 is a poorly characterized RNA-binding RING E3-ubiquitin ligase with functions in embryonic development. Here we demonstrate that DZIP3 is a crucial driver of cancer cell growth, migration, and invasion. In mice and zebrafish cancer models, DZIP3 promoted tumor growth and metastasis. In line with these results, DZIP3 was frequently overexpressed in several cancer types. Depletion of DZIP3 from cells resulted in reduced expression of Cyclin D1 and a subsequent G1 arrest and defect in cell growth. Mechanistically, DZIP3 utilized its two different domains to interact and stabilize Cyclin D1 both at mRNA and protein levels. Using an RNA-binding lysine-rich region, DZIP3 interacted with the AU-rich region in 3' untranslated region of Cyclin D1 mRNA and stabilized it. Using a RING E3-ligase domain, DZIP3 interacted and increased K63-linked ubiquitination of Cyclin D1 protein to stabilize it. Remarkably, DZIP3 interacted with, ubiquitinated, and stabilized Cyclin D1 predominantly in the G1 phase of the cell cycle, where it is needed for cell-cycle progression. In agreement with this, a strong positive correlation of mRNA expression between DZIP3 and Cyclin D1 in different cancer types was observed. Additionally, DZIP3 regulated several cell cycle proteins by modulating the Cyclin D1-E2F axes. Taken together, this study demonstrates for the first time that DZIP3 uses a unique two-pronged mechanism in its stabilization of Cyclin D1 to drive cell-cycle and cancer progression. SIGNIFICANCE: These findings show that DZIP3 is a novel driver of cell-cycle and cancer progression via its control of Cyclin D1 mRNA and protein stability in a cell-cycle phase-dependent manner. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/315/F1.large.jpg.

Although hormonal therapy (HT) inhibits the growth of hormone receptor-positive (HR+) breast and prostate cancers, HT resistance frequently develops within the complex metastatic microenvironment of the host organ (often the bone), a setting poorly recapitulated in 2D culture systems. To address this limitation, we cultured HR+ breast cancer and prostate cancer spheroids and patient-derived organoids in 3D extracellular matrices (ECM) alone or together with bone marrow stromal cells (BMSC). In 3D monocultures, antiestrogens and antiandrogens induced anoikis by abrogating anchorage-independent growth of HR+ cancer cells but exhibited only modest effects against tumor cells residing in the ECM niche. In contrast, BMSC induced hormone-independent growth of breast cancer and prostate cancer spheroids and restored lumen filling in the presence of HR-targeting agents. Molecular and functional characterization of BMSC-induced hormone independence and HT resistance in anchorage-independent cells revealed distinct context-dependent mechanisms. Cocultures of ZR75-1 and LNCaP with BMSCs exhibited paracrine IL6-induced HT resistance via attenuation of HR protein expression, which was reversed by inhibition of IL6 or JAK signaling. Paracrine IL6/JAK/STAT3-mediated HT resistance was confirmed in patient-derived organoids cocultured with BMSCs. Distinctly, MCF7 and T47D spheroids retained ER protein expression in cocultures but acquired redundant compensatory signals enabling anchorage independence via ERK and PI3K bypass cascades activated in a non-IL6-dependent manner. Collectively, these data characterize the pleiotropic hormone-independent mechanisms underlying acquisition and restoration of anchorage-independent growth in HR+ tumors. Combined analysis of tumor and microenvironmental biomarkers in metastatic biopsies of HT-resistant patients can help refine treatment approaches. SIGNIFICANCE: This study uncovers a previously underappreciated dependency of tumor cells on HR signaling for anchorage-independent growth and highlights how the metastatic microenvironment restores this malignant property of cancer cells during hormone therapy.

Comment on Cancer Res. 2020 Dec 1;80(23):5367-5379.

Alteration in lipid composition is an important metabolic adaptation by cancer cells to support tumorigenesis and metastasis. Fatty acid 2-hydroxylase (FA2H) introduces a chiral hydroxyl group at the second carbon of fatty acid (FA) backbones and influences lipid structures and metabolic signaling. However, the underlying mechanisms through which FA 2-hydroxylation is coupled to metabolic adaptation and tumor growth remain elusive. Here, we show that FA2H regulates specific metabolic reprogramming and oncogenic signaling in the development of colorectal cancer. FA2H is highly expressed in normal colorectal tissues. Assessments through deciphering both published high-throughput data and curated human colorectal cancer samples revealed significant suppression of FA2H in tumors, which is correlated with unfavorable prognosis. Experiments with multiple models of genetic manipulation or treatment with an enzymatic product of FA2H, (R)-2-hydroxy palmitic acid, demonstrated that FA 2-hydroxylation inhibits colorectal cancer cell proliferation, migration, epithelial-to-mesenchymal transition progression, and tumor growth. Bioinformatics analysis suggested that FA2H functions through AMP-activated protein kinase/Yes-associated protein (AMPK/YAP) pathway, which was confirmed in colorectal cancer cells, as well as in tumors. Lipidomics analysis revealed an accumulation of polyunsaturated fatty acids in cells with FA2H overexpression, which may contribute to the observed nutrient deficiency and AMPK activation. Collectively, these data demonstrate that FA 2-hydroxylation initiates a metabolic signaling cascade to suppress colorectal tumor growth and metastasis via the YAP transcriptional axis and provides a strategy to improve colorectal cancer treatment. SIGNIFICANCE: These findings identify a novel metabolic mechanism regulating the tumor suppressor function of FA 2-hydroxylation in colorectal cancer.

Aberrant Wnt signaling drives a number of cancers through regulation of diverse downstream pathways. Wnt/β-catenin signaling achieves this in part by increasing the expression of proto-oncogenes such as MYC and cyclins. However, global assessment of the Wnt-regulated transcriptome in vivo in genetically distinct cancers demonstrates that Wnt signaling suppresses the expression of as many genes as it activates. In this study, we examined the set of genes that are upregulated upon inhibition of Wnt signaling in Wnt-addicted pancreatic and colorectal cancer models. Decreasing Wnt signaling led to a marked increase in gene expression by activating ERK and JNK; these changes in gene expression could be mitigated in part by concurrent inhibition of MEK. These findings demonstrate that increased Wnt signaling in cancer represses MAPK activity, preventing RAS-mediated senescence while allowing cancer cells to proliferate. These results shift the paradigm from Wnt/β-catenin primarily as an activator of transcription to a more nuanced view where Wnt/β-catenin signaling drives both widespread gene repression and activation. SIGNIFICANCE: These findings show that Wnt/β-catenin signaling causes widespread gene repression via inhibition of MAPK signaling, thus fine tuning the RAS-MAPK pathway to optimize proliferation in cancer.

Circular RNAs (circRNA) are a new member of endogenously produced noncoding RNAs that have been characterized as key regulators of gene expression in a variety of malignances. However, the role of circRNA in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we identified unique circRNA that regulate OSCC progression and metastasis and pave roads for future research in early diagnosis, prevention, and treatment of OSCC. Transcriptomic analyses identified a circRNA derived from IGHG locus (circIGHG) as significantly upregulated in OSCC and positively associated with poor prognosis of OSCC. circIGHG directly bound miR-142-5p and consequently elevated IGF2BP3 activity. Knockdown of circIGHG led to impaired expression of IGF2BP3 and attenuated aggressiveness of OSCC cells. Epithelial-mesenchymal transition was the main mechanism through which circIGHG/IGF2BP3 promotes metastasis of OSCC. Overall, these results demonstrate that circIGHG plays a pivotal role in OSCC development and metastasis and has potential to serve as a biomarker and therapeutic target for early-stage diagnosis and treatment of OSCC. SIGNIFICANCE: These findings broaden our insights regarding regulation of OSCC progression by circular RNA and serve as a reference for future clinical research in OSCC diagnosis and treatment.

Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7- cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7-β-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential "persister cancer cell" phenotype. SIGNIFICANCE: Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.

Although neoadjuvant chemotherapy is a standard component of breast cancer treatment, recent evidence suggests that chemotherapeutic drugs can promote metastasis through poorly defined mechanisms. Here we utilize xenograft mouse models of triple-negative breast cancer to explore the importance of chemotherapy-induced tumor-derived small extracellular vesicles (sEV) in metastasis. Doxorubicin (DXR) enhanced tumor cell sEV secretion to accelerate pulmonary metastasis by priming the premetastatic niche. Proteomic analysis and CRISPR/Cas9 gene editing identified the inflammatory glycoprotein PTX3 enriched in DXR-elicited sEV as a critical regulator of chemotherapy-induced metastasis. Both genetic inhibition of sEV secretion from primary tumors and pharmacologic inhibition of sEV uptake in secondary organs suppressed metastasis following chemotherapy. Taken together, this research uncovers a mechanism of chemotherapy-mediated metastasis by which drug-induced upregulation of sEV secretion and PTX3 protein cargo primes the premetastatic niche and suggests that inhibition of either sEV uptake in secondary organs or secretion from primary tumor cells may be promising therapeutic strategies to suppress metastasis. SIGNIFICANCE: These findings show that chemotherapy-induced small extracellular vesicles accelerate breast cancer metastasis, and targeted inhibition of tumor-derived vesicles may be a promising therapeutic strategy to improve the efficacy of chemotherapy treatment.

Ras activates its effectors at the membrane. Active PI3Kα and its associated kinases/phosphatases assemble at membrane regions enriched in signaling lipids. In contrast, the Raf kinase domain extends into the cytoplasm and its assembly is away from the crowded membrane surface. Our structural membrane-centric outlook underscores the spatiotemporal principles of membrane and signaling lipids, which helps clarify PI3Kα activation. Here we focus on mechanisms of activation driven by PI3Kα driver mutations, spotlighting the PI3Kα double (multiple) activating mutations. Single mutations can be potent, but double mutations are stronger: their combination is specific, a single strong driver cannot fully activate PI3K, and two weak drivers may or may not do so. In contrast, two strong drivers may successfully activate PI3K, where one, for example, H1047R, modulates membrane interactions facilitating substrate binding at the active site (km) and the other, for example, E542K and E545K, reduces the transition state barrier (ka), releasing autoinhibition by nSH2. Although mostly unidentified, weak drivers are expected to be common, so we ask here how common double mutations are likely to be and why PI3Kα with double mutations responds effectively to inhibitors. We provide a structural view of hotspot and weak driver mutations in PI3Kα activation, explain their mechanisms, compare these with mechanisms of Raf activation, and point to targeting cell-specific, chromatin-accessible, and parallel (or redundant) pathways to thwart the expected emergence of drug resistance. Collectively, our biophysical outlook delineates activation and highlights the challenges of drug resistance.

The transcription factor E74-like factor 3 (ELF3) is inactivated in a range of cancers, including biliary tract cancer (BTC). Here, we investigated the tumor-suppressive role of ELF3 in bile duct cells by identifying several previously unknown direct target genes of ELF3 that appear to be implicated in biliary duct carcinogenesis. ELF3 directly repressed ZEB2, a key regulator of epithelial-mesenchymal transition, and upregulated the expression of CGN, an integral element in lumen formation. Loss of ELF3 led to decreased cell-cell junctions and enhanced cell motility. ALOX5 and CXCL16 were also identified as additional direct targets of ELF3; their corresponding proteins 5-lipoxygenase and CXCL16 play a role in the immune response. Conditioned medium from cells overexpressing ELF3 significantly enhanced the migration of natural killer cells and CD8+ T cells toward the conditioned medium. Gene expression profiling for BTC expressing high levels of ELF3 revealed significant enrichment of the ELF3-related genes. In a BTC xenograft model, activation of ELF3 increased expression of ELF3-related genes, enhanced the tubular structure of the tumors, and led to a loss of vimentin. Overall, our results indicate that ELF3 is a key regulator of both epithelial integrity and immune responses in BTC. SIGNIFICANCE: Thease finding shows that ELF3 regulates epithelial integrity and host immune responses and functions as a tumor suppressor in biliary tract cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/489/F1.large.jpg.

The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer. SIGNIFICANCE: This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.

Tumors are complex tissues composed of transformed epithelial cells as well as cancer-activated fibroblasts (CAF) that facilitate epithelial tumor cell invasion. We show here that CAFs and other mesenchymal cells rely much more on glutamine than epithelial tumor cells; consequently, they are more sensitive to inhibition of glutaminase. Glutamine dependence drove CAF migration toward this amino acid when cultured in low glutamine conditions. CAFs also invaded a Matrigel matrix following a glutamine concentration gradient and enhanced the invasion of tumor cells when both cells were cocultured. Accordingly, glutamine directed invasion of xenografted tumors in immunocompromised mice. Stimulation of glutamine-driven epithelial tumor invasion by fibroblasts required previous CAF activation, which involved the TGFβ/Snail1 signaling axis. CAFs moving toward Gln presented a polarized Akt2 distribution that was modulated by the Gln-dependent activity of TRAF6 and p62 in the migrating front, and depletion of these proteins prevented Akt2 polarization and Gln-driven CAF invasion. Our results demonstrate that glutamine deprivation promotes CAF migration and invasion, which in turn facilitates the movement of tumor epithelial cells toward nutrient-rich territories. These results provide a novel molecular mechanism for how metabolic stress enhances invasion and metastasis. SIGNIFICANCE: Cancer-associated fibroblasts migrate and invade toward free glutamine and facilitate invasion of tumor epithelial cells, accounting for their movement away from the hostile conditions of the tumor towards nutrient-rich adjacent tissues. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/438/F1.large.jpg.

Cellular dedifferentiation is a key mechanism driving cancer progression. Acquisition of mesenchymal features has been associated with drug resistance, poor prognosis, and disease relapse in many tumor types. Therefore, successful targeting of tumors harboring these characteristics is a priority in oncology practice. The SWItch/Sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has also emerged as a critical player in tumor progression, leading to the identification of several SWI/SNF complex genes as potential disease biomarkers and targets of anticancer therapies. AT-rich interaction domain-containing protein 1A (ARID1A) is a component of SWI/SNF, and mutations in ARID1A represent one of the most frequent molecular alterations in human cancers. ARID1A mutations occur in approximately 10% of pancreatic ductal adenocarcinomas (PDAC), but whether these mutations confer a therapeutic opportunity remains unclear. Here, we demonstrate that loss of ARID1A promotes an epithelial-mesenchymal transition (EMT) phenotype and sensitizes PDAC cells to a clinical inhibitor of HSP90, NVP-AUY922, both in vitro and in vivo. Although loss of ARID1A alone did not significantly affect proliferative potential or rate of apoptosis, ARID1A-deficient cells were sensitized to HSP90 inhibition, potentially by promoting the degradation of intermediate filaments driving EMT, resulting in cell death. Our results describe a mechanistic link between ARID1A defects and a quasi-mesenchymal phenotype, suggesting that deleterious mutations in ARID1A associated with protein loss exhibit potential as a biomarker for patients with PDAC who may benefit by HSP90-targeting drugs treatment. SIGNIFICANCE: This study identifies ARID1A loss as a promising biomarker for the identification of PDAC tumors that are potentially responsive to treatment with proteotoxic agents.

Although next-generation sequencing is widely used in cancer to profile tumors and detect variants, most somatic variant callers used in these pipelines identify variants at the lowest possible granularity, single-nucleotide variants (SNV). As a result, multiple adjacent SNVs are called individually instead of as a multi-nucleotide variants (MNV). With this approach, the amino acid change from the individual SNV within a codon could be different from the amino acid change based on the MNV that results from combining SNV, leading to incorrect conclusions about the downstream effects of the variants. Here, we analyzed 10,383 variant call files (VCF) from the Cancer Genome Atlas (TCGA) and found 12,141 incorrectly annotated MNVs. Analysis of seven commonly mutated genes from 178 studies in cBioPortal revealed that MNVs were consistently missed in 20 of these studies, whereas they were correctly annotated in 15 more recent studies. At the BRAF V600 locus, the most common example of MNV, several public datasets reported separate BRAF V600E and BRAF V600M variants instead of a single merged V600K variant. VCFs from the TCGA Mutect2 caller were used to develop a solution to merge SNV to MNV. Our custom script used the phasing information from the SNV VCF and determined whether SNVs were at the same codon and needed to be merged into MNV before variant annotation. This study shows that institutions performing NGS sequencing for cancer genomics should incorporate the step of merging MNV as a best practice in their pipelines. SIGNIFICANCE: Identification of incorrect mutation calls in TCGA, including clinically relevant BRAF V600 and KRAS G12, will influence research and potentially clinical decisions.

ATM kinase is a tumor suppressor and a master regulator of the DNA damage response. Most cancer-associated alterations to ATM are missense mutations at the PI3-kinase regulatory domain (PRD) or the kinase domain. Expression of kinase-dead (KD) ATM protein solely accelerates lymphomagenesis beyond ATM loss. To understand how PRD suppresses lymphomagenesis, we introduced the cancer-associated PRD mutation R3008H (R3016 in mouse) into mice. R3008H abrogated DNA damage- and oxidative stress-induced activation of ATM without consistently affecting ATM protein stability and recruitment. In contrast to the early embryonic lethality of AtmKD/KD mice, AtmR3016H (AtmR/R ) mice were viable, immunodeficient, and displayed spontaneous craniofacial abnormalities and delayed lymphomagenesis compared with Atm-/- controls. Mechanistically, R3008H rescued the tardy exchange of ATM-KD at DNA damage foci, indicating that PRD coordinates ATM activation with its exchange at DNA-breaks. Taken together, our results reveal a unique tumorigenesis profile for PRD mutations that is distinct from null or KD mutations. SIGNIFICANT: This study functionally characterizes the most common ATM missense mutation R3008H in cancer and identifies a unique role of PI3-kinase regulatory domain in ATM activation.

Biomechanics is a fundamental feature of a cell. However, the manner by which actomysin tension affects tumor immune evasion remains unclear. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill soft tumor-repopulating cells (TRC). TRC softness prevented membrane pore formation caused by CTL-released perforin. Perforin interacting with nonmuscle myosin heavy-chain 9 transmitted forces to less F-actins in soft TRC, thus generating an inadequate contractile force for perforin pore formation. Stiffening TRC allowed perforin the ability to drill through the membrane, leading to CTL-mediated killing of TRC. Importantly, overcoming mechanical softness in human TRC also enhanced TRC cell death caused by human CTL, potentiating a mechanics-based immunotherapeutic strategy. These findings reveal a mechanics-mediated tumor immune evasion, thus potentially providing an alternative approach for tumor immunotherapy. SIGNIFICANCE: Tumor-repopulating cells evade CD8+ cytolytic T-cell killing through a mechanical softness mechanism, underlying the impediment of perforin pore formation at the immune synapse site.

The ability of a receptor to preferentially activate only a subset of available downstream signal cascades is termed biased signaling. Although comprehensively recognized for the G protein-coupled receptors (GPCR), this process is scarcely explored downstream of receptor tyrosine kinases (RTK), including the cancer-relevant insulin-like growth factor-1 receptor (IGF1R). Successful IGF1R targeting requires receptor downregulation, yet therapy-mediated removal from the cell surface activates cancer-protective β-arrestin-biased signaling (β-arr-BS). As these overlapping processes are initiated by the β-arr/IGF1R interaction and controlled by GPCR-kinases (GRK), we explored GRKs as potential anticancer therapeutic targets to disconnect IGF1R downregulation and β-arr-BS. Transgenic modulation demonstrated that GRK2 inhibition or GRK6 overexpression enhanced degradation of IGF1R, but both scenarios sustained IGF1-induced β-arr-BS. Pharmacologic inhibition of GRK2 by the clinically approved antidepressant, serotonin reuptake inhibitor paroxetine (PX), recapitulated the effects of GRK2 silencing with dose- and time-dependent IGF1R downregulation without associated β-arr-BS. In vivo, PX treatment caused substantial downregulation of IGF1R, suppressing the growth of Ewing's sarcoma xenografts. Functional studies reveal that PX exploits the antagonism between β-arrestin isoforms; in low ligand conditions, PX favored β-arrestin1/Mdm2-mediated ubiquitination/degradation of IGF1R, a scenario usually exclusive to ligand abundancy, making PX more effective than antibody-mediated IGF1R downregulation. This study provides the rationale, molecular mechanism, and validation of a clinically feasible concept for "system bias" targeting of the IGF1R to uncouple downregulation from signaling. Demonstrating system bias as an effective anticancer approach, our study reveals a novel strategy for the rational design or repurposing of therapeutics to selectively cross-target the IGF1R or other RTK. SIGNIFICANCE: This work provides insight into the molecular and biological roles of biased signaling downstream RTK and provides a novel "system bias" strategy to increase the efficacy of anti-IGF1R-targeted therapy in cancer.

Invasive lobular breast carcinoma (ILC), one of the major breast cancer histologic subtypes, exhibits unique features compared with the well-studied ductal cancer subtype (IDC). The pathognomonic feature of ILC is loss of E-cadherin, mainly caused by inactivating mutations, but the contribution of this genetic alteration to ILC-specific molecular characteristics remains largely understudied. To profile these features transcriptionally, we conducted single-cell RNA sequencing on a panel of IDC and ILC cell lines, and an IDC cell line (T47D) with CRISPR-Cas9-mediated E-cadherin knockout (KO). Inspection of intracell line heterogeneity illustrated genetically and transcriptionally distinct subpopulations in multiple cell lines and highlighted rare populations of MCF7 cells highly expressing an apoptosis-related signature, positively correlated with a preadaptation signature to estrogen deprivation. Investigation of E-cadherin KO-induced alterations showed transcriptomic membranous systems remodeling, elevated resemblance to ILCs in regulon activation, and increased sensitivity to IFNγ-mediated growth inhibition via activation of IRF1. This study reveals single-cell transcriptional heterogeneity in breast cancer cell lines and provides a resource to identify drivers of cancer progression and drug resistance. SIGNIFICANCE: This study represents a key step towards understanding heterogeneity in cancer cell lines and the role of E-cadherin depletion in contributing to the molecular features of invasive lobular breast carcinoma.

Investigating metabolic rewiring in cancer can lead to the discovery of new treatment strategies for breast cancer subtypes that currently lack targeted therapies. In this study, we used MMTV-Myc-driven tumors to model breast cancer heterogeneity, investigating the metabolic differences between two histologic subtypes, the epithelial-mesenchymal transition (EMT) and the papillary subtypes. A combination of genomic and metabolomic techniques identified differences in nucleotide metabolism between EMT and papillary subtypes. EMT tumors preferentially used the nucleotide salvage pathway, whereas papillary tumors preferred de novo nucleotide biosynthesis. CRISPR/Cas9 gene editing and mass spectrometry-based methods revealed that targeting the preferred pathway in each subtype resulted in greater metabolic impact than targeting the nonpreferred pathway. Knocking out the preferred nucleotide pathway in each subtype has a deleterious effect on in vivo tumor growth, whereas knocking out the nonpreferred pathway has a lesser effect or may even result in increased tumor growth. Collectively, these data suggest that significant differences in metabolic pathway utilization distinguish EMT and papillary subtypes of breast cancer and identify said pathways as a means to enhance subtype-specific diagnoses and treatment strategies. SIGNIFICANCE: These findings uncover differences in nucleotide salvage and de novo biosynthesis using a histologically heterogeneous breast cancer model, highlighting metabolic vulnerabilities in these pathways as promising targets for breast cancer subtypes.

In the last decade, large-scale genomic studies in patients with hematologic malignancies identified recurrent somatic alterations in epigenetic modifier genes. Among these, the de novo DNA methyltransferase DNMT3A has emerged as one of the most frequently mutated genes in adult myeloid as well as lymphoid malignancies and in clonal hematopoiesis. In this review, we discuss recent advances in our understanding of the biochemical and structural consequences of DNMT3A mutations on DNA methylation catalysis and binding interactions and summarize their effects on epigenetic patterns and gene expression changes implicated in the pathogenesis of hematologic malignancies. We then review the role played by mutant DNMT3A in clonal hematopoiesis, accompanied by its effect on immune cell function and inflammatory responses. Finally, we discuss how this knowledge informs therapeutic approaches for hematologic malignancies with mutant DNMT3A.

Approximately 70% of all breast cancers are estrogen receptor-positive (ER+ breast cancer), and endocrine therapy has improved survival for patients with ER+ breast cancer. However, up to half of these tumors recur within 20 years. Recurrent ER+ breast cancers develop resistance to endocrine therapy; thus, novel targets are needed to treat recurrent ER+ breast cancer. Here we report that semaphorin 7A (SEMA7A) confers significantly decreased patient survival rates in ER+ breast cancer. SEMA7A was hormonally regulated in ER+ breast cancer, but its expression did not uniformly decrease with antiestrogen treatments. Additionally, overexpression of SEMA7A in ER+ cell lines drove increased in vitro growth in the presence of estrogen deprivation, tamoxifen, and fulvestrant. In vivo, SEMA7A conferred primary tumor resistance to fulvestrant and induced lung metastases. Prosurvival signaling was identified as a therapeutic vulnerability of ER+SEMA7A+ tumors. We therefore propose that targeting this pathway with inhibitors of survival signaling such as venetoclax may prove efficacious for treating SEMA7A+ tumors. SIGNIFICANCE: SEMA7A predicts for and likely contributes to poor response to standard-of-care therapies, suggesting that patients with SEMA7A+ER+ tumors may benefit from alternative therapeutic strategies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/1/187/F1.large.jpg.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease that is associated with increased risk of lung cancer. Pseudomonas aeruginosa (PA) infections are frequent in patients with COPD, which increase lung inflammation and acute exacerbations. However, the influences of PA-induced inflammation on lung tumorigenesis and the efficacy of immune checkpoint blockade remain unknown. In this study, we initiated a murine model of lung cancer by treating FVB/NJ female mice with tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alone or in combination with PA-lipopolysaccharide (LPS). LPS-mediated chronic inflammation induced T-cell exhaustion, increased the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, and enhanced NNK-induced lung tumorigenesis through an immunosuppressive microenvironment characterized by accumulation of myeloid-derived suppressive cells (MDSC) and regulatory T cells. Anti-PD-1 antibody treatment reduced tumors in NNK/LPS-treated mice with a 10-week LPS treatment but failed to inhibit tumor growth when LPS exposure was prolonged to 16 weeks. Anti-Ly6G antibody treatment coupled with depletion of MDSC alone reduced tumor growth; when combined with anti-PD-1 antibody, this treatment further enhanced antitumor activity in 16-week NNK/LPS-treated mice. Immune gene signatures from a human lung cancer dataset of PD-1 blockade were identified, which predicted treatment responses and survival outcome and overlapped with those from the mouse model. This study demonstrated that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti-PD-1 treatment in mice. Immune gene signatures overlap with human and mouse lung tumors, providing potentially predictive markers for patients undergoing immunotherapy. SIGNIFICANCE: This study identifies an immune gene signature that predicts treatment responses and survival in patients with tobacco carcinogen-induced lung cancer receiving immune checkpoint blockade therapy.

Comment in J Urol. 2021 Sep;206(3):775-776.

Comment in Cancer Res. 2021 Jan 1;81(1):35-37.

Notch activation has been detected in pancreatic ductal adenocarcinoma (PDAC). However, its role in PDAC metastasis remains unknown. In this study, we identify a Notch-dependent feedback circuit between pancreatic cancer cells and macrophages, which contributes to PDAC metastasis. In this circuit, miR-124 regulated Notch signaling in cancer cells by directly targeting the Notch ligand Jagged 1. Autoamplified Notch signaling promoted the recruitment and activation of macrophages to a tumor-supporting M2-like phenotype via downstream IL8, CCL2, IL1α, and uPA paracrine signaling. In turn, activated macrophage-derived IL6 activated the oncogenic transcription factor STAT3 that directly repressed miR-124 genes via a conserved STAT3-binding site in their promoters, thereby promoting cancer cell epithelial-mesenchymal transition and invasion. Disrupting this circuit suppressed liver metastasis in mouse models. Thus, our study suggests that manipulation of this Notch-dependent circuit has a therapeutic potential for the treatment of PDAC metastasis. SIGNIFICANCE: This study provided potential therapeutic targets and robust preclinical evidence for PDAC treatment by interrupting feedback signaling between cancer cells and macrophages with targeted inhibitors.

Glioma stem cells (GSC) are a subpopulation of tumor cells with special abilities to proliferate and differentiate in gliomas. They are one of the main causes of tumor recurrence, especially under hypoxic conditions. Although long noncoding RNAs (lncRNA) are known to be involved in numerous biological processes and are implied in the occurrence of certain diseases, their role in tumor development and progression remains poorly understood. Here we explored the mechanisms by which lncRNA derived from hypoxic GSCs (H-GSC) cause glioma progression. Isolation and identification of the Linc01060 gene, the exosomes containing them, and the proteins from tumor cells regulating the gene allowed for studying the effects of Linc01060 on proliferation and glycometabolism. H-GSC exerted their effects by transferring exosomes to glioma cells, resulting in a significant increase in Linc01060 levels. Mechanistically, Linc01060 directly interacted with the transcription factor myeloid zinc finger 1 (MZF1) and enhanced its stability. Linc01060 facilitated nuclear translocation of MZF1 and promoted MZF1-mediated c-Myc transcriptional activities. In addition, c-Myc enhanced the accumulation of the hypoxia-inducible factor-1 alpha (HIF1α) at the posttranscriptional level. HIF1α bound the hormone response elements of the Linc01060 promoter, upregulating the transcription of Linc01060 gene. Clinically, Linc01060 was upregulated in glioma and was significantly correlated with tumor grade and poor clinical prognosis. Overall, these data show that secretion of Linc01060-containing exosomes from H-GSCs activates prooncogenic signaling pathways in glioma cells to promote disease progression. SIGNIFICANCE: These findings suggest that inhibition of Linc01060-containing exosomes or targeting the Linc01060/MZF1/c-Myc/HIF1α axis may be an effective therapeutic strategy in glioma.

Comment in Cancer Res. 2021 Jan 1;81(1):129-143.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. SIGNIFICANCE: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone Apc Min/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APC Min offspring mice were largely free of intestinal adenoma, and several chromosome substitution (consomic) strains carrying single PWD chromosomes on the B6 genetic background displayed reduced adenoma numbers. Multiple dosage-dependent modifier loci on PWD chromosome 5 each contributed to tumor suppression. Activation of β-catenin-driven and stem cell-specific gene expression in the presence of Apc Min or following APC loss remained moderate in intestines carrying PWD chromosome 5, suggesting that PWD variants restrict adenoma initiation by controlling stem cell homeostasis. Gene expression of modifier candidates and DNA methylation on chromosome 5 were predominantly cis controlled and largely reflected parental patterns, providing a genetic basis for inheritance of tumor susceptibility. Human SNP variants of several modifier candidates were depleted in colorectal cancer genomes, suggesting that similar mechanisms may also affect the penetrance of cancer driver mutations in humans. Overall, our analysis highlights the strong impact that multiple genetic variants acting in networks can exert on tumor development. SIGNIFICANCE: These findings in mice show that, in addition to accidental mutations, cancer risk is determined by networks of individual gene variants.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvβ5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that de novo expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.

Lipid rafts are tightly packed, cholesterol- and sphingolipid-enriched microdomains within the plasma membrane that play important roles in many pathophysiologic processes. Rafts have been strongly implicated as master regulators of signal transduction in cancer, where raft compartmentalization can promote transmembrane receptor oligomerization, shield proteins from enzymatic degradation, and act as scaffolds to enhance intracellular signaling cascades. Cancer cells have been found to exploit these mechanisms to initiate oncogenic signaling and promote tumor progression. This review highlights the roles of lipid rafts within the metastatic cascade, specifically within tumor angiogenesis, cell adhesion, migration, epithelial-to-mesenchymal transition, and transendothelial migration. In addition, the interplay between lipid rafts and different modes of cancer cell death, including necrosis, apoptosis, and anoikis, will be described. The clinical role of lipid raft-specific proteins, caveolin and flotillin, in assessing patient prognosis and evaluating metastatic potential of various cancers will be presented. Collectively, elucidation of the complex roles of lipid rafts and raft components within the metastatic cascade may be instrumental for therapeutic discovery to curb prometastatic processes.

Tumor-derived secretory factors orchestrate splenic hematopoietic and stromal cells to fuel metastasis. The spleen acts as a reservoir site for hematopoietic stem and progenitor cells, which are rapidly exploited as myeloid-derived suppressor cells at the cost of tumor-reactive lymphoid cells. Splenic erythroid progenitor cells and mesenchymal stromal cells contribute directly and indirectly to both tumor immune escape and the metastatic cascade. Animal models provide valuable mechanistic insights, but their translation to a clinical setting highlights specific challenges and open issues. In this review, we envision the exploitation of the spleen as a source for novel biomarkers and therapeutic approaches.

Cyclin-dependent kinase 12 (CDK12) is a member of the CDK family of proteins (CDK) and is critical for cancer development. Years of study into CDK12 have generated much information regarding the intricacy of its function and mechanism as well as inhibitors against it for oncological research. However, there remains a lack of understanding regarding the role of CDK12 in carcinogenesis and cancer prevention. An exhaustive comprehension of CDK12 will highly stimulate the development of new strategies for treating and preventing cancer. Here, we review the literature of CDK12, with a focus on its function, its role in signaling, and how to use it as a target for discovery of novel drugs for cancer prevention and therapy.

Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft-tissue sarcoma. Multimodal treatment, including surgery and traditional chemotherapy with radiotherapy, has contributed to improvements in overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here, we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models in vitro and in vivo resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small-molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant antitumor and antimetastatic potential in several primary and metastatic in vivo models, including a relapsed RMS patient-derived xenograft model. Transcriptomic analysis of PAK4-targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of antitumor immune response signatures. This PAK4-targeting gene signature showed prognostic significance for patients with sarcoma. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS. SIGNIFICANCE: These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced rhabdomyosarcoma.

HOTAIR is a lncRNA overexpressed in several epithelial cancers and strongly correlated with invasion. This lncRNA was proven a pivotal element of the epithelial-to-mesenchymal transition (EMT), a transdifferentiation process triggering metastasis. Snail, master inducer of EMT, requires HOTAIR to recruit EZH2 on specific epithelial target genes (i.e., HNF4α, E-cadherin, and HNF1α) and cause their repression. Here, we designed a HOTAIR deletion mutant form, named HOTAIR-sbid, including the putative Snail-binding domain but depleted of the EZH2-binding domain. HOTAIR-sbid acted as a dominant negative of the endogenous HOTAIR. In both murine and human tumor cells, HOTAIR-sbid impaired the ability of HOTAIR to bind Snail and, in turn, trigger H3K27me3/EZH2-mediated repression of Snail epithelial target genes. Notably, HOTAIR-sbid expression was proven to reduce cellular motility, invasiveness, anchorage-independent growth, and responsiveness to TGFβ-induced EMT. These data provide evidence on a lncRNA-based strategy to effectively impair the function of a master EMT-transcriptional factor. SIGNIFICANCE: This study defines an innovative RNA-based strategy to interfere with a pivotal function of the tumor-related lncRNA HOTAIR, comprising a dominant negative mutant that was computationally designed and that impairs epithelial-to-mesenchymal transition.

Emerging studies indicate that DNA damage in cancer cells triggers antitumor immunity, but its intrinsic regulatory mechanism in breast cancer cells remains poorly understood. Here, we show that ZMYND8 is upregulated and inhibits micronucleus formation and DNA damage in breast cancer cells. Loss of ZMYND8 triggered activation of the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase in micronuclei, leading to further activation of the downstream signaling effectors stimulator of IFN genes and NF-κB, but not TANK-binding kinase 1 and IFN regulatory factor 3, thereby inducing the expression of IFNβ and IFN-stimulated genes (ISG) in breast cancer cells in vitro and tumors in vivo. ZMYND8 knockout (KO) in breast cancer cells promoted infiltration of CD4+ and CD8+ T cells, leading to tumor inhibition in syngeneic mouse models, which was significantly attenuated by treatment of anti-CD4/CD8-depleting antibodies or anti-IFNAR1 antibody and in immunodeficient Rag1 KO mice. In human breast tumors, ZMYND8 was negatively correlated with ISGs, CD4, CD8A, CD8B, and the tumor-lymphocyte infiltration phenotype. Collectively, these findings demonstrate that maintenance of genome stability by ZMYND8 causes breast cancer cells to evade cytotoxic T-lymphocyte surveillance, which leads to tumor growth. SIGNIFICANCE: These findings show that ZMYND8 is a new negative and intrinsic regulator of the innate immune response in breast tumor cells, and ZMYND8 may be a possible target for antitumor immunotherapy.

Recent years have seen the emergence of conditionally activated diagnostics and therapeutics that leverage protease-cleavable peptide linkers to enhance their specificity for cancer. However, due to a lack of methods to measure and localize protease activity directly within the tissue microenvironment, the design of protease-activated agents has been necessarily empirical, yielding suboptimal results when translated to patients. To address the need for spatially resolved protease activity profiling in cancer, we developed a new class of in situ probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZP) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. AZPs were leveraged within a generalizable framework to design conditional cancer diagnostics and therapeutics and showcased in the Hi-Myc mouse model of prostate cancer, which models features of early pathogenesis. Multiplexed screening against barcoded substrates yielded a peptide, S16, that was robustly and specifically cleaved by tumor-associated metalloproteinases in the Hi-Myc model. In situ labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, premalignant Hi-Myc prostatic glands. Systemic administration of an in vivo imaging probe incorporating S16 perfectly classified diseased and healthy prostates, supporting the relevance of ex vivo activity assays to in vivo translation. We envision AZPs will enable new insights into the biology of protease dysregulation in cancer and accelerate the development of conditional diagnostics and therapeutics for multiple cancer types. SIGNIFICANCE: Visualization of protease activity within the native tissue context using AZPs provides new biological insights into protease dysregulation in cancer and guides the design of conditional diagnostics and therapeutics.

Histone methyltransferase NSD3 is frequently dysregulated in human cancers, yet the epigenetic role of NSD3 during cancer development remains elusive. Here we report that NSD3-induced methylation of H3K36 is crucial for breast tumor initiation and metastasis. In patients with breast cancer, elevated expression of NSD3 was associated with recurrence, distant metastasis, and poor survival. In vivo, NSD3 promoted malignant transformation of mammary epithelial cells, a function comparable to that of HRAS. Furthermore, NSD3 expanded breast cancer-initiating cells and promoted epithelial-mesenchymal transition to trigger tumor invasion and metastasis. Mechanistically, the long isoform (full-length transcript) of NSD3, but not its shorter isoform lacking a catalytic domain, cooperated with EZH2 and RNA polymerase II to stimulate H3K36me2/3-dependent transactivation of genes associated with NOTCH receptor cleavage, leading to nuclear accumulation of NICD and NICD-mediated transcriptional repression of E-cadherin. Furthermore, mice harboring primary and metastatic breast tumors with overexpressed NSD3 showed sensitivity to NOTCH inhibition. Together, our findings uncover the critical epigenetic role of NSD3 in the modulation of NOTCH-dependent breast tumor progression, providing a rationale for targeting the NSD3-NOTCH signaling regulatory axis in aggressive breast cancer. SIGNIFICANCE: This study demonstrates the functional significance of histone methyltransferase NSD3 in epigenetic regulation of breast cancer stemness, EMT, and metastasis, suggesting NSD3 as an actionable therapeutic target in metastatic breast cancer.