Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.

Transcription factor ZIC2 regulates the tumorigenic phenotypes associated with both bulk and cancer stem cells in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America. Current therapeutic regimens are ineffective against advanced EOC. A better understanding of the molecular mechanisms that regulate the biology of EOC will be a critical step toward developing more efficacious therapies against EOC. Herein, we demonstrate that elevated expression of transcription factor ZIC2 was associated with lower survival of EOC patients. Knockout of endogenous ZIC2 in EOC cells attenuated the tumorigenic phenotypes associated with both bulk and cancer stem cells in vitro and in vivo, indicating a pro-tumorigenic role of ZIC2 in EOC. On the other hand, however, overexpression of ZIC2 in EOC cells that do not express endogenous ZIC2 promoted cell migration and sphere formation, but inhibited cell growth and colony formation in vitro and tumor growth in vivo, indicating that the role for ZIC2 in EOC is context dependent. Our transcriptomic analysis showed that ZIC2-regulated genes were involved in multiple biological processes and signaling pathways associated with tumor progression. In conclusion, our findings reveal a context-dependent role for ZIC2 in regulating tumorigenic phenotypes in EOC, providing evidence that ZIC2 can be a potential therapeutic target for EOCs that express a high level of ZIC2.

MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors.

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR), which is a pivotal metabolic regulator, and epigenetic modifications remains poorly understood. Our results show that mTORC1 activation caused by the abrogation of its negative regulator tuberous sclerosis complex 2 (TSC2) coincides with increased levels of the histone modification H3K27me3 but not H3K4me3 or H3K9me3. This selective H3K27me3 induction was mediated via 4E-BP-dependent increase in EZH2 protein levels. Surprisingly, mTOR inhibition also selectively induced H3K27me3. This was independent of TSC2, and was paralleled by reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the induction of H3K27me3 may potentiate the anti-proliferative effects of mTOR inhibitors.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

Melanoma is the leading cause of skin cancer-related death. As prognosis of patients with melanoma remains problematic, identification of new therapeutic targets remains essential. Matricellular proteins are nonstructural extracellular matrix proteins. They are secreted into the tumor microenvironment to coordinate behavior among different cell types, yet their contribution to melanoma is underinvestigated. Examples of matricellular proteins include those comprising the CCN family. The CCN family member, CCN1, is highly proangiogenic. Herein, we show that, in human patients with melanoma, although found in several tumor cell types, CCN1 is highly expressed by a subset of cancer-associated fibroblasts (CAF) in patients with melanoma and this expression correlates positively with expression of proangiogenic genes and progressive disease/resistance to anti-PD1 checkpoint inhibitors. Consistent with these observations, in a syngeneic C57BL6 mouse model of melanoma, loss of CCN1 expression from Col1A2-Cre-, herein identified as "universal," fibroblasts, impaired metastasis of subcutaneously injected B16F10 tumor cells to lung, concomitant with disrupted neovascularization and collagen organization. Disruption of the extracellular matrix in the loss of CCN1 was validated using a novel artificial intelligence-based image analysis platform that revealed significantly decreased phenotypic fibrosis and composite morphometric collagen scores. As drug resistance is linked to matrix deposition and neoangiogenesis, these data suggest that CCN1, due to its multifaceted role, may represent a novel therapeutic target for drug-resistant melanoma. Our data further emphasize the essential role that cancer-associated, (universal) Col1A2-Cre-fibroblasts and extracellular matrix remodeling play in coordinating behavior among different cell types within the tumor microenvironment. SIGNIFICANCE: In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Wounding the stroma: Docetaxel's role in dormant breast cancer escape.

The mechanistic underpinnings of breast cancer recurrence following periods of dormancy are largely undetermined. A new study in PLOS Biology reveals that docetaxel-induced injury of tumour stromal cells stimulates the release of cytokines that support dormancy escape of breast cancer cells.

Establishment and validation of preclinical models of SMARCA4-inactivated and ARID1A/ARID1B co-inactivated dedifferentiated endometrial carcinoma.

OBJECTIVE: Dedifferentiated endometrial cancer (DDEC) is an uncommon and clinically highly aggressive subtype of endometrial cancer characterized by genomic inactivation of SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protein. It responds poorly to conventional systemic treatment and its rapidly progressive clinical course limits the therapeutic windows to trial additional lines of therapies. This underscores a pressing need for biologically accurate preclinical tumor models to accelerate therapeutic development. METHODS: DDEC tumor from surgical samples were implanted into immunocompromised mice for patient-derived xenograft (PDX) and cell line development. The histologic, immunophenotypic, genetic and epigenetic features of the patient tumors and the established PDX models were characterized. The SMARCA4-deficienct DDEC model was evaluated for its sensitivity toward a KDM6A/B inhibitor (GSK-J4) that was previously reported to be effective therapy for other SMARCA4-deficient cancer types. RESULTS: All three DDEC models exhibited rapid growth in vitro and in vivo, with two PDX models showing spontaneous development of metastases in vivo. The PDX tumors maintained the same undifferentiated histology and immunophenotype, and exhibited identical genomic and methylation profiles as seen in the respective parental tumors, including a mismatch repair (MMR)-deficient DDEC with genomic inactivation of SMARCA4, and two MMR-deficient DDECs with genomic inactivation of both ARID1A and ARID1B. Although the SMARCA4-deficient cell line showed low micromolecular sensitivity to GSK-J4, no significant tumor growth inhibition was observed in the corresponding PDX model. CONCLUSIONS: These established patient tumor-derived models accurately depict DDEC and represent valuable preclinical tools to gain therapeutic insights into this aggressive tumor type.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Phosphorylation of eIF4E in the stroma drives the production and spatial organisation of collagen type I in the mammary gland.

The extracellular matrix (ECM) plays critical roles in breast cancer development. Whether ECM composition is regulated by the phosphorylation of eIF4E on serine 209, an event required for tumorigenesis, has not been explored. Herein, we used proteomics and mouse modeling to investigate the impact of mutating serine 209 to alanine on eIF4E (i.e., S209A) on mammary gland (MG) ECM. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028953. We discovered that S209A knock-in mice, expressing a non-phosphorylatable form of eIF4E, have less collagen-I deposition in native and tumor-bearing MGs, leading to altered tumor cell invasion. Additionally, phospho-eIF4E deficiency impacts collagen topology; fibers at the tumor-stroma boundary in phospho-eIF4E-deficient mice run parallel to the tumor edge but radiate outwards in wild-type mice. Finally, a phospho-eIF4E-deficient tumor microenvironment resists anti-PD-1 therapy-induced collagen deposition, correlating with an increased anti-tumor response to immunotherapy. Clinically, we showed that collagen-I and phospho-eIF4E are positively correlated in human breast cancer samples, and that stromal phospho-eIF4E expression is influenced by tumor proximity. Together, our work defines the importance of phosphorylation of eIF4E on S209 as a regulator of MG collagen architecture in the tumor microenvironment, thereby positioning phospho-eIF4E as a therapeutic target to augment response to therapy.

Docosahexaenoic acid enrichment of tumor phospholipid membranes increases tumor necroptosis in mice bearing triple negative breast cancer patient-derived xenografts.

Docosahexaenoic acid (DHA) reduces breast cancer tumor growth in preclinical models. To better understand how DHA amplifies the actions of docetaxel (TXT) chemotherapy, we examined the effects of two doses of dietary DHA on tumor size, membrane DHA content and necroptosis using a drug resistant triple negative breast cancer (TNBC) patient derived xenograft (PDX) model. Female NOD.Cb-PrkdcscidIl2rg mice bearing TNBC PDXs were randomized to one of three nutritionally complete diets (20% w/w fat): control (0% DHA), high DHA (3.8% HDHA), or low DHA (1.6% LDHA) with or without intraperitoneal injections of 5 mg/kg TXT, twice weekly for 6 weeks (n=8 per group). Tumors from mice fed either HDHA+TXT or LDHA+TXT were similar in size to each other, but were 36% and 32% smaller than tumors from mice fed control+TXT, respectively (P<.05). A dose effect of DHA incorporation was observed in plasma total phospholipids and in phosphatidylethanolamine and phosphatidylinositol. Both doses of DHA resulted in similarly increased necrotic tissue and decreased NFκB protein expression compared to control tumors, however only the HDHA+TXT had increased expression of necroptosis related proteins: RIPK1, RIPK3 and MLKL (P<.05). Increased MLKL was observed in the lipid raft portion of HDHA+TXT tumor extracts. This work confirms the efficacy of a combination therapy consisting of DHA supplementation and TXT chemotherapy using two doses of DHA as indicated by reduced tumor growth in a TNBC PDX model. Moreover, the results suggest that decreased growth may occur through increased DHA incorporation into tumor phospholipid membranes and necroptosis.

Docosahexaenoic Acid in the Inhibition of Tumor Cell Growth in Preclinical Models of Ovarian Cancer.

There is a strong rationale for investigating nutritional interventions with docosahexaenoic acid (DHA) in cancer prevention and therapy; however, the effects of DHA on ovarian cancer (OC) have not been well studied. Here, we investigated if DHA alone and in combination with carboplatin reduces OC cell growth in vitro. In vivo, we used a high-grade serous OC patient-derived xenograft (PDX) mouse model to investigate if DHA affects OC growth and enhances the anticancer actions of carboplatin. We showed synergistic cell killing by DHA and carboplatin in DHA-resistant Kuramochi and SKOV3 OC cells, which corresponded with increased DHA incorporation into whole-cell membrane phospholipids (P < 0.05). In vivo, feeding mice a diet supplemented with 3.9% (w/w of fat) DHA resulted in a significant reduction in PDX growth with and without carboplatin (P < 0.05). This reduction in tumor growth was accompanied by an increased tumor necrotic region (P < 0.05) and improved survival. Plasma membranes in tumors and livers excised from mice fed a DHA diet had ∼ twofold increase in DHA incorporation as compared with mice fed a control diet. Our findings indicate that DHA supplementation reduces cancer cell growth and enhances the efficacy of carboplatin in preclinical models of OC through increased apoptosis and necrosis.Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1952453.

Genome profiles of pathologist-defined cell clusters by multiregional LCM and G&T-seq in one triple-negative breast cancer patient.

Pathological examination is the gold standard for cancer diagnosis, and breast tumor cells are often found in clusters. We report a case study on one triple-negative breast cancer (TNBC) patient, analyzing tumor development, metastasis, and prognosis with simultaneous DNA and RNA sequencing of pathologist-defined cell clusters from multiregional frozen sections. The cell clusters are isolated by laser capture microdissection (LCM) from primary tumor tissue, lymphatic vessels, and axillary lymph nodes. Data are reported for a total of 97 cell clusters. A combination of tumor cell-cluster clonality and phylogeny reveals 3 evolutionarily distinct pathways for this patient, each associated with a unique mRNA signature, and each correlated with disparate survival outcomes. Hub gene analysis indicates that extensive downregulation of ribosomal protein mRNA is a potential marker of poor prognosis in breast cancer.

Characterization of ovarian cancer-derived extracellular vesicles by surface-enhanced Raman spectroscopy.

Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop methods for the early diagnosis of ovarian cancer. A step towards early and non-invasive cancer diagnosis is through the utilization of extracellular vesicles (EVs), which are nanoscale, membrane-bound vesicles that contain proteins and genetic material reflective of their parent cell. Thus, EVs secreted by cancer cells can be thought of as cancer biomarkers. In this paper, we present gold nanohole arrays for the capture of ovarian cancer (OvCa)-derived EVs and their characterization by surface-enhanced Raman spectroscopy (SERS). For the first time, we have characterized EVs isolated from two established OvCa cell lines (OV-90, OVCAR3), two primary OvCa cell lines (EOC6, EOC18), and one human immortalized ovarian surface epithelial cell line (hIOSE) by SERS. We subsequently determined their main compositional differences by principal component analysis and were able to discriminate the groups by a logistic regression-based machine learning method with ∼99% accuracy, sensitivity, and specificity. The results presented here are a great step towards quick, facile, and non-invasive cancer diagnosis.

Robust inflammatory breast cancer gene signature using nonparametric random forest analysis.

Inflammatory breast cancer (IBC) is a rare, aggressive cancer found in all the molecular breast cancer subtypes. Despite extensive previous efforts to screen for transcriptional differences between IBC and non-IBC patients, a robust IBC-specific molecular signature has been elusive. We report a novel IBC-specific gene signature (59 genes; G59) that achieves 100% accuracy in discovery and validation samples (45/45 correct classification) and remarkably only misclassified one sample (60/61 correct classification) in an independent dataset. G59 is independent of ER/HER2 status, molecular subtypes and is specific to untreated IBC samples, with most of the genes being enriched for plasma membrane cellular component proteins, interleukin (IL), and chemokine signaling pathways. Our finding suggests the existence of an IBC-specific molecular signature, paving the way for the identification and validation of targetable genomic drivers of IBC.

SMARCA4/2 loss inhibits chemotherapy-induced apoptosis by restricting IP3R3-mediated Ca2+ flux to mitochondria.

Inactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca2+) release in these cancers. By restricting chromatin accessibility to ITPR3, encoding Ca2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca2+-dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers.

Genetically-encoded discovery of proteolytically stable bicyclic inhibitors for morphogen NODAL.

In this manuscript, we developed a two-fold symmetric linchpin (TSL) that converts readily available phage-displayed peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where X is any amino acid but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma cells. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-TYK-nu ovarian carcinoma cells with apparent IC50 of 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control TYK-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (Pronase™) for 21 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides to expand the previously reported repertoire of phage-displayed bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

N-3 Long-Chain Polyunsaturated Fatty Acids, Eicosapentaenoic and Docosahexaenoic Acid, and the Role of Supplementation during Cancer Treatment: A Scoping Review of Current Clinical Evidence.

This scoping review examines the evidence for n-3 long-chain polyunsaturated fatty acid [LCPUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] supplementation in clinical cancer therapy. A comprehensive literature search was performed to identify relevant clinical intervention studies conducted through August 2020. Fifty-seven unique cancer trials, assessing EPA and/or DHA supplementation pre- or post-treatment, concomitant with neoadjuvant chemotherapy, radiation or surgery, or in palliative therapy were included. Breast, head and neck, gastrointestinal, gastric, colorectal/rectal, esophageal, leukemia/lymphoma, lung, multiple myeloma and pancreatic cancers were investigated. Across the spectrum of cancers, the evidence suggests that supplementation increased or maintained body weight, increased progression-free and overall survival, improved overall quality of life, resulted in beneficial change in immune parameters and decreased serious adverse events. Taken together, the data support that EPA and/or DHA could be used to improve outcomes important to the patient and disease process. However, before incorporation into treatment can occur, there is a need for randomized clinical trials to determine the dose and type of n-3 LCPUFA intervention required, and expansion of outcomes assessed and improved reporting of outcomes.