Combined Inhibition of SHP2 and CXCR1/2 Promotes Antitumor T-cell Response in NSCLC.

SHP2 inhibitors (SHP2i) alone and in various combinations are being tested in multiple tumors with overactivation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages, induced tumor-intrinsic CCL5/CXCL10 secretion, and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non-small cell lung cancer (NSCLC). However, treatment also increased intratumor granulocytic myeloid-derived suppressor cells (gMDSC) via tumor-intrinsic, NFκB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C inhibitor trials. Combined SHP2 (SHP099)/CXCR1/2 (SX682) inhibition depleted a specific cluster of S100a8/9 hi gMDSCs, generated Klrg1 + CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras- and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in patients with NSCLC. SIGNIFICANCE: Our study shows that inhibiting the SHP2/RAS/ERK pathway triggers NFκB-dependent upregulation of CXCR2 ligands and recruitment of S100A8hi gMDSCs, which suppress T cells. Combining SHP2/CXCR2 inhibitors blocks gMDSC immigration, resulting in enhanced Th1 polarization, induced CD8+KLRG1+ effector T cells with high cytotoxic activity, and improved survival in multiple NSCLC models.This article is highlighted in the In This Issue feature, p. 1.

Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer.

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTP), a subpopulation of cancer cells that survive via reversible, nongenetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKI) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single-cell RNA sequencing reveals that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting shows that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation. SIGNIFICANCE: DTPs are implicated in resistance to anticancer therapies, but their ontogeny and vulnerabilities remain unclear. We find that HER2 TKI-DTPs emerge from stochastically arising primed cells ("pre-DTPs") that engage either of two distinct transcriptional programs upon TKI exposure. Our results provide new insights into DTP ontogeny and potential therapeutic vulnerabilities. This article is highlighted in the In This Issue feature, p. 873.

Dysregulated Kras/YY1/ZNF322A/Shh transcriptional axis enhances neo-angiogenesis to promote lung cancer progression.

Angiogenesis enhances cancer metastasis and progression, however, the roles of transcription regulation in angiogenesis are not fully defined. ZNF322A is an oncogenic zinc-finger transcription factor. Here, we demonstrate a new mechanism of Kras mutation-driven ZNF322A transcriptional activation and elucidate the interplay between ZNF322A and its upstream transcriptional regulators and downstream transcriptional targets in promoting neo-angiogenesis. Methods: Luciferase activity, RT-qPCR and ChIP-qPCR assays were used to examine transcription regulation in cell models. In vitro and in vivo angiogenesis assays were conducted. Immunohistochemistry, Kaplan-Meier method and multivariate Cox regression assays were performed to examine the clinical correlation in tumor specimens from lung cancer patients. Results: We validated that Yin Yang 1 (YY1) upregulated ZNF322A expression through targeting its promoter in the context of Kras mutation. Reconstitution experiments by knocking down YY1 under KrasG13V activation decreased KrasG13V-promoted cancer cell migration, proliferation and ZNF322A promoter activity. Knockdown of YY1 or ZNF322A attenuated angiogenesis in vitro and in vivo. Notably, we validated that ZNF322A upregulated the expression of sonic hedgehog (Shh) gene which encodes a secreted factor that activates pro-angiogenic responses in endothelial cells. Clinically, ZNF322A protein expression positively correlated with Shh and CD31, an endothelial cell marker, in 133 lung cancer patient samples determined using immunohistochemistry analysis. Notably, patients with concordantly high expression of ZNF322A, Shh and CD31 correlated with poor prognosis. Conclusions: These findings highlight the mechanism by which dysregulation of Kras/YY1/ZNF322/Shh transcriptional axis enhances neo-angiogenesis and cancer progression in lung cancer. Therapeutic strategies that target Kras/YY1/ZNF322A/Shh signaling axis may provide new insight on targeted therapy for lung cancer patients.

Oncogenic zinc finger protein ZNF322A promotes stem cell-like properties in lung cancer through transcriptional suppression of c-Myc expression.

ZNF322A, a C2H2 zinc finger transcription factor, is an oncoprotein in lung cancer. However, the transcription mechanisms of ZNF322A in lung cancer stem cell-like reprogramming remain elusive. By integrating our chromatin immunoprecipitation-sequencing and RNA-sequencing datasets, we identified and validated the transcriptional targets of ZNF322A, which were significantly enriched in tumorigenic functions and developmental processes. Indeed, overexpression of ZNF322A promoted self-renewal ability and increased stemness-related gene expressions in vitro and in vivo. Importantly, ZNF322A bound directly to c-Myc promoter and recruited histone deacetylase 3 to transcriptionally suppress c-Myc expression, which in turn increased mitochondrial oxidative phosphorylation and promoted cell motility, thus maintaining stem cell-like properties of lung cancer. Clinically, ZNF322AHigh/c-MycLow expression profile was revealed as an independent indicator of poor prognosis in lung cancer patients. Our study provides the first evidence that ZNF322A-centered transcriptome promotes lung tumorigenesis and ZNF322A acts as a transcription suppressor of c-Myc to maintain lung cancer stem cell-like properties by shifting metabolism towards oxidative phosphorylation.

SHP2 Inhibition Prevents Adaptive Resistance to MEK Inhibitors in Multiple Cancer Models.

Adaptive resistance to MEK inhibitors (MEKi) typically occurs via induction of genes for different receptor tyrosine kinases (RTK) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 (PTPN11) is required for RAS/ERK pathway activation by most RTKs and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEKi inhibited the proliferation of multiple cancer cell lines in vitro PTPN11 knockdown/MEKi treatment had similar effects, whereas expressing SHP099 binding-defective PTPN11 mutants conferred resistance, demonstrating that SHP099 is on-target. SHP099/trametinib was highly efficacious in xenograft and/or genetically engineered models of KRAS-mutant pancreas, lung, and ovarian cancers and in wild-type RAS-expressing triple-negative breast cancer. SHP099 inhibited activation of KRAS mutants with residual GTPase activity, impeded SOS/RAS/MEK/ERK1/2 reactivation in response to MEKi, and blocked ERK1/2-dependent transcriptional programs. We conclude that SHP099/MEKi combinations could have therapeutic utility in multiple malignancies.Significance: MEK inhibitors show limited efficacy as single agents, in part because of the rapid development of adaptive resistance. We find that SHP2/MEK inhibitor combinations prevent adaptive resistance in multiple cancer models expressing mutant and wild-type KRAS. Cancer Discov; 8(10); 1237-49. ©2018 AACR. See related commentary by Torres-Ayuso and Brognard, p. 1210 This article is highlighted in the In This Issue feature, p. 1195.

Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment.

The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4-containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.

Oct4 transcriptionally regulates the expression of long non-coding RNAs NEAT1 and MALAT1 to promote lung cancer progression.

BACKGROUND: Oct4, a key stemness transcription factor, is overexpressed in lung cancer. Here, we reveal a novel transcription regulation of long non-coding RNAs (lncRNAs) by Oct4. LncRNAs have emerged as important players in cancer progression. METHODS: Oct4 chromatin-immunoprecipitation (ChIP)-sequencing and several lncRNA databases with literature annotation were integrated to identify Oct4-regulated lncRNAs. Luciferase activity, qRT-PCR and ChIP-PCR assays were conducted to examine transcription regulation of lncRNAs by Oct4. Reconstitution experiments of Oct4 and downstream lncRNAs in cell proliferation, migration and invasion assays were performed to confirm the Oct4-lncRNAs signaling axes in promoting lung cancer cell growth and motility. The expression correlations between Oct4 and lncRNAs were investigated in 124 lung cancer patients using qRT-PCR analysis. The clinical significance of Oct4/lncRNAs signaling axes were further evaluated using multivariate Cox regression and Kaplan-Meier analyses. RESULTS: We confirmed that seven lncRNAs were upregulated by direct binding of Oct4. Among them, nuclear paraspeckle assembly transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and urothelial carcinoma-associated 1 (UCA1) were validated as Oct4 transcriptional targets through promoter or enhancer activation. We showed that lung cancer cells overexpressing NEAT1 or MALAT1 and the Oct4-silenced cells reconstituted with NEAT1 or MALAT1 promoted cell proliferation, migration and invasion. In addition, knockdown of NEAT1 or MALAT1 abolished Oct4-mediated lung cancer cell growth and motility. These cell-based results suggested that Oct4/NEAT1 or Oct4/MALAT1 axis promoted oncogenesis. Clinically, Oct4/NEAT1/MALAT1 co-overexpression was an independent factor for prediction of poor outcome in 124 lung cancer patients. CONCLUSIONS: Our study reveals a novel mechanism by which Oct4 transcriptionally activates NEAT1 via promoter and MALAT1 via enhancer binding to promote cell proliferation and motility, and led to lung tumorigenesis and poor prognosis.

A histone deacetylase inhibitor enhances expression of genes inhibiting Wnt pathway and augments activity of DNA demethylation reagent against nonsmall-cell lung cancer.

Development of new inhibitors targeting histone deacetylases (HDACs) with improved efficacy for solid tumor therapy is urgently needed. Here, we report the development of a novel HDAC inhibitor TMU-35435 and verify it as a single agent and in combination treatment with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza-dC) in lung cancer preclinical models. TMU-35435 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. Expression microarrays revealed a unique TMU-35435-induced gene networks enriched in biological processes, including "negative regulation of cell proliferation" and "Wnt receptor signaling pathway" compared to FDA-approved HDAC inhibitor SAHA. TMU-35435 inhibited tumor growth with good pharmacokinetic properties and safety features in lung orthotopic and subcutaneously implanted xenograft models. TMU-35435 and 5-aza-dC showed synergistic antitumor effects through reactivation of tumor suppressor genes and those genes encoding negative regulators of Wnt signaling pathway in vitro and in vivo. Some genes showed additive inhibition of DNA methylation upon TMU-35435 and 5-aza-dC combined treatment. Our findings suggested that TMU-35435 is a potential HDAC inhibitor for lung cancer treatment as a single agent and in combination with 5-aza-dC.

A prognostic predictor panel with DNA methylation biomarkers for early-stage lung adenocarcinoma in Asian and Caucasian populations.

BACKGROUND: The incidence of lung adenocarcinoma (LUAD) is increasing worldwide with different prognosis even in early-stage patients. We aimed to identify a prognostic panel with multiple DNA methylation biomarkers to predict survival in early-stage LUAD patients of different racial groups. METHODS: The methylation array, pyrosequencing methylation assay, Cox regression and Kaplan-Meier analyses were conducted to build the risk score equations of selected probes in a training cohort of 69 Asian LUAD patients. The risk score model was verified in another cohort of 299 Caucasian LUAD patients in The Cancer Genome Atlas (TCGA) database. RESULTS: We performed a Cox regression analysis, in which the regression coefficients were obtained for eight probes corresponding to eight genes (AGTRL1, ALDH1A3, BDKRB1, CTSE, EFNA2, NFAM1, SEMA4A and TMEM129). The risk score was derived from sum of each methylated probes multiplied by its corresponding coefficient. Patients with the risk score greater than the median value showed poorer overall survival compared with other patients (p = 0.007). Such a risk score significantly predicted patients showing poor survival in TCGA cohort (p = 0.036). A multivariate analysis was further performed to demonstrate that the eight-probe panel association with poor outcome in early-stage LUAD patients remained significant even after adjusting for different clinical variables including staging parameters (hazard ratio, 2.03; p = 0.039). CONCLUSIONS: We established a proof-of-concept prognostic panel consisting of eight-probe signature to predict survival of early-stage LUAD patients of Asian and Caucasian populations.

Zinc finger proteins in cancer progression.

Zinc finger proteins are the largest transcription factor family in human genome. The diverse combinations and functions of zinc finger motifs make zinc finger proteins versatile in biological processes, including development, differentiation, metabolism and autophagy. Over the last few decades, increasing evidence reveals the potential roles of zinc finger proteins in cancer progression. However, the underlying mechanisms of zinc finger proteins in cancer progression vary in different cancer types and even in the same cancer type under different types of stress. Here, we discuss general mechanisms of zinc finger proteins in transcription regulation and summarize recent studies on zinc finger proteins in cancer progression. In this review, we also emphasize the importance of further investigations in elucidating the underlying mechanisms of zinc finger proteins in cancer progression.

Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis.

Rab small GTPases are master regulators of membrane trafficking and guide vesicle targeting. Recent publications show that Rab-controlled trafficking pathways are altered during tumorigenesis. However, whether any of the Rabs plays a metastasis suppressor role is least explored. Here we address the metastasis suppressive function of human Rab37 (hRAB37) using secretomics, cell, animal and clinical analyses. We show that tissue inhibitor of metalloproteinase 1 (TIMP1), a secreted glycoprotein that inhibits extracellular matrix turnover, is a novel cargo of hRAB37. hRAB37 regulates the exocytosis of TIMP1 in a nucleotide-dependent manner to inactivate matrix metalloproteinase 9 (MMP9) migration axis in vitro and in vivo. Dysfunction of hRAB37 or TIMP1 abrogates metastasis suppression. Lung cancer patients with metastasis and poor survival show low hRAB37 protein expression coinciding with low TIMP1 in tumours. Our findings identify hRAB37 as a novel metastasis suppressor Rab that functions through the TIMP1-MMP9 pathway and has significant prognostic power.