RNA Sequencing Reveals Novel Oncogenic Fusions and Depicts Detailed Fusion Transcripts of FN1-FGFR1 in Phosphaturic Mesenchymal Tumors.

Phosphaturic mesenchymal tumors (PMTs) are rare neoplasms of soft tissue or bone. Although previous studies revealed that approximately 50% of PMTs harbor FN1::FGFR1 fusions, the molecular mechanisms in the remaining cases are largely unknown. In this study, fusion genes were investigated using RNA-based next-generation sequencing in 76 retrospectively collected PMTs. Novel fusions were validated with Sanger sequencing and fluorescence in situ hybridization. Fusion genes were detected in 52/76 (68.4%) PMTs, and 43/76 (56.6%) harbored FN1::FGFR1 fusions. Fusion transcripts and breakpoints of the FN1::FGFR1 fusions were diverse. The most common fusion transcript was between exon 20 of FN1 and exon 9 of FGFR1 (7/43, 16.3%). The most upstream breakpoint of the FN1 gene was located at the 3' end of exon 12, and the most downstream breakpoint of the FGFR1 gene was at the 5' end of exon 9, suggesting the inessential nature of the third fibronectin-type domain of FN1 and the necessity of the transmembrane domain of FGFR1 in the FN1::FGFR1 fusion protein, respectively. Moreover, the reciprocal FGFR1::FN1 fusions, which had not been identified in previous studies, were detected in 18.6% (8/43) of FN1::FGFR1 fusion-positive PMTs. Novel fusions were identified in 6/76 (7.9%) FN1::FGFR1 fusion-negative PMTs, including 2 involving FGFR: FGFR1::USP33 (1/76, 1.3%) and FGFR1::TLN1 (1/76, 1.3%). Other novel fusions identified were the PDGFRA::USP35 (1/76, 1.3%), SPTBN1::YWHAQ (1/76, 1.3%), GTF2I::RALGPS1 (1/76, 1.3%), and LTBP1::VWA8 (1/76, 1.3%) fusions. In addition to these novel fusions, FN1::FGFR2 (1/76, 1.3%), NIPBL::BEND2 (1/76, 1.3%), and KIAA1549::BRAF fusions (1/76, 1.3%) were also identified in FN1::FGFR1-negative cases arising from the thigh, ilium, and acetabulum, respectively. The frequency of oncogenic fusions was significantly higher (P = .012) in tumors derived from extremities (29/35, 82.9%) compared with other locations (23/41, 56.1%). No significant correlation was identified between fusions and recurrence (P = .786). In conclusion, we report fusion transcripts and breakpoints of FN1::FGFR1 in PMTs in detail, providing insights into fusion protein functions. We also revealed that a considerable proportion of PMTs without FN1::FGFR1 fusion carried novel fusions, providing further insight into the genetic basis of PMTs.

Molecular and Cytogenetic Features of NTRK Fusions Enriched in BRAF and RET Double-Negative Papillary Thyroid Cancer.

Rare NTRK-driven malignant neoplasms can be effectively inhibited by anti-TRK agents. The discovery of NTRK1/2/3-rich tumors in papillary thyroid cancer (PTC) patients is a precondition for the rapid identification of NTRK fusion tumors. Knowledge of NTRK gene activation is critical to accurately detect NTRK status. A total of 229 BRAF V600E-negative samples from PTC patients were analyzed in this study. Break-apart fluorescence in situ hybridization (FISH) was performed to detect RET fusion. NTRK status was analyzed using FISH, DNA- and RNA-based next-generation sequencing, and quantitative reverse transcription PCR. In 128 BRAF and RET double-negative cases, 56 (43.8%, 56/128) NTRK rearrangement tumors were found, including 1 NTRK2, 16 NTRK1, and 39 NTRK3 fusions. Two novel NTRK fusions, EZR::NTRK1 and EML4::NTRK2, were found in the NTRK rearrangement tumors. Dominant break-apart and extra 3' signal patterns accounted for 89.3% (50/56) and 5.4% (3/56) of all NTRK-positive cases, respectively, as determined by FISH. In this study's cohort, there were 2.3% (3/128) FISH false-negative and 3.1% (4/128) FISH false-positive cases identified. NTRK fusions are highly recurrent in BRAF and RET double-negative PTCs. FISH- or RNA-based next-generation sequencing is a reliable detection approach. NTRK rearrangement can be precisely, rapidly, and economically detected based on the developed optimal algorithm.

NF-κB represses retinoic acid receptor-mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia.

Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulation plays a critical role. Lung tumor suppressor G protein-coupled receptor, family C, member 5A (GPRC5A), is repressed in most non-small cell lung cancer (NSCLC); however, the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induced GPRC5A repression both in vitro and in vivo. Intriguingly, transactivation of NF-κB downstream targets was not required, but the transactivation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in the GPRC5A promoter. Instead, p65 was complexed with retinoic acid receptor α/ß (RARα/ß) and recruited to the RA response element site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complexing and suppressed transcription. Notably, phosphorylation on serine 276 of p65 was required for interaction with RARα/ß and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression was through suppression of acetylated histone H3K9 (H3K9ac), but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a histone deacetylase inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via a complex with RARα/ß and mediates epigenetic repression via suppression of H3K9ac.

Identification of Active Bronchioalveolar Stem Cells as the Cell of Origin in Lung Adenocarcinoma.

While initiation is established as a critical step in tumorigenesis, the identity of the cell of origin for lung adenocarcinoma and the mechanism controlling susceptibility to initiation remain elusive. Here we show that lung tumor suppressor Gprc5a-knockout (KO) mice are susceptible to initiation of lung tumorigenesis. Bronchioalveolar stem cells (BASC) and alveolar type 2 (AT2) cells were aberrantly expanded in Gprc5a-KO mouse lungs compared with those in wild-type (WT) mice, suggesting that Gprc5a-KO might confer susceptibility to initiation by increasing the cell of origin in mouse lungs. BASCs from Gprc5a-KO mice (KO-BASC) exhibited significantly increased stemness and self-renewal potential and reduced differentiation capacity compared with BASCs from WT mice (WT-BASC). AT2 cells did not possess self-renewal potential regardless of Gprc5a status. KO-BASCs expressed a stem-like gene profile with upregulated Abcg2, EGFR, and NF-κB signaling compared with WT-BASCs. Blockade of EGFR and NF-κB signaling inhibited both expansion of BASC and AT2 cells and lung tumorigenesis. Abcg2 was expressed in active KO-BASCs as well as in lung tumor cells but not in quiescent WT-BASCs or AT2 cells, supporting that lung adenocarcinoma cells are derived from Abcg2-positive KO-BASCs (active). Taken together, Gprc5a deletion leads to expansion of active BASCs via dysregulated EGFR and NF-κB signaling that confers susceptibility to initiation of lung tumorigenesis, marking Abcg2-positive BASCs as candidate cell of origin for lung adenocarcinoma. SIGNIFICANCE: Identification of active bronchioalveolar stem cells as lung adenocarcinoma cells of origin provides insights into mechanisms of lung tumorigenesis and could facilitate development of effective strategies for cancer prevention and therapy. See related commentary by Osborne and Minna, p. 972.

PD-L1, Mismatch Repair Protein, and NTRK Immunohistochemical Expression in Cervical Small Cell Neuroendocrine Carcinoma.

BACKGROUND: Cervical small cell neuroendocrine carcinoma (SCNC) is a rare and aggressive disease that lacks a standard treatment strategy or effective methods of targeted therapy. PD-L1 inhibitors for DNA mismatch repair system-deficient (dMMR) tumors and neurotrophin receptor tyrosine kinase (NTRK) inhibitors offer potential pan-cancer treatments. METHODS: Immunohistochemistry was employed as the main detection method, and any NTRK positive cases, identified by immunohistochemistry, were further submitted for evaluation by fluorescence in situ hybridization (FISH) and real-time polymerase chain reaction (RT-PCR) methods. RESULTS: Forty-six patients were enrolled. Positive PD-L1 expression was seen in 22 of the 43 patients (51.16%) with an average combined positive score of 6.82. PD-L1-positive patients were more likely to have a higher proliferation rate in the tumor, and they experienced less recurrence and death (p = 0.048 and 0.033, respectively) compared with the patients with negative PD-L1 expression. However, in the multivariate analysis, none of the clinical parameters was associated with the expression of PD-L1. There was no association between PD-L1 expression and disease recurrence or overall survival in the Kaplan-Meier analysis. All cases were found to be MMR-stable and lacked NTRK gene fusion. However, pan-Trk expressed in 14 (32.56%) of the 43 tested cases, but FISH and RT-PCR failed to confirm any positive fusion signals in IHC-positive cases. CONCLUSIONS: PD-L1 may be an effective therapeutic target for cervical SCNC. Cervical SCNC is a MMR-stable tumor and lacks NTRK gene fusion. IHC isn't a reliable method in the detection of NTRK gene fusion in cervical SCNC.

Evaluation of NTRK Gene Fusion by Five Different Platforms in Triple-Negative Breast Carcinoma.

Triple-negative breast carcinoma (TNBC) is an aggressive disease that has a poor prognosis since it lacks effective treatment methods. Neurotrophic tyrosine receptor kinase (NTRK) fusion genes are excellent candidates for targeted RTK inhibitor therapies and there are available targeted therapy drugs for the treatment of TRK fusion-positive tumors in a tumor agnostic pattern. Our study was designed to investigate the NTRK gene fusion status in TNBC patients and to determine whether RTK-targeted therapies are suitable for TNBC patients. A total of 305 TNBC patients were enrolled in our study. IHC was employed as a prescreening method, and IHC positive cases were further submitted for evaluation by FISH, RT-PCR, and NGS methods. NTRK IHC was evaluated successfully in 287 of the 305 cases, and there were 32 (11.15%) positive cases. FISH was carried out in the 32 IHC positive cases. There were 13 FISH-positive cases if the threshold was set as >15% of the 100 counted tumor cells having a split orange and green signal with more than one signal diameter. There were only 2 FISH-positive cases if the cutoff value was defined as >15% of the counted tumor cells having a split signal with more than two signal diameter widths. One of the FISH-positive cases had a separate NTRK3 FISH signal in 88% of the tumor cells, and its IHC result was strong nuclear staining in all the tumor cells. After evaluation of the morphology, it was re-diagnosed as secretory breast carcinoma, and the NGS result confirmed that it had a NTRK3-ETV6 fusion gene. The other FISH-positive cases were all negative for NTRK gene fusion in the NGS or RT-PCR examination. The NTRK gene fusion rate was low in our TNBC cohort. NTRK gene fusion may be a rare event in TNBC. The high false-positive rate of NTRK gene fusion detected by IHC questions its role as a prescreening method in TNBC. More data may be needed to determine a suitable threshold for NTRK FISH in TNBC in the future. More studies are needed to confirm whether RTK-targeted therapies are appropriate treatments for TNBC patients.

Dysregulated Glutamate Transporter SLC1A1 Propels Cystine Uptake via Xc- for Glutathione Synthesis in Lung Cancer.

Cancer cells need to generate large amounts of glutathione (GSH) to buffer oxidative stress during tumor development. A rate-limiting step for GSH biosynthesis is cystine uptake via a cystine/glutamate antiporter Xc-. Xc- is a sodium-independent antiporter passively driven by concentration gradients from extracellular cystine and intracellular glutamate across the cell membrane. Increased uptake of cystine via Xc- in cancer cells increases the level of extracellular glutamate, which would subsequently restrain cystine uptake via Xc-. Cancer cells must therefore evolve a mechanism to overcome this negative feedback regulation. In this study, we report that glutamate transporters, in particular SLC1A1, are tightly intertwined with cystine uptake and GSH biosynthesis in lung cancer cells. Dysregulated SLC1A1, a sodium-dependent glutamate carrier, actively recycled extracellular glutamate into cells, which enhanced the efficiency of cystine uptake via Xc- and GSH biosynthesis as measured by stable isotope-assisted metabolomics. Conversely, depletion of glutamate transporter SLC1A1 increased extracellular glutamate, which inhibited cystine uptake, blocked GSH synthesis, and induced oxidative stress-mediated cell death or growth inhibition. Moreover, glutamate transporters were frequently upregulated in tissue samples of patients with non-small cell lung cancer. Taken together, active uptake of glutamate via SLC1A1 propels cystine uptake via Xc- for GSH biosynthesis in lung tumorigenesis. SIGNIFICANCE: Cellular GSH in cancer cells is not only determined by upregulated Xc- but also by dysregulated glutamate transporters, which provide additional targets for therapeutic intervention.

Hexokinase 2 Depletion Confers Sensitization to Metformin and Inhibits Glycolysis in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinomas (SCCs) are highly aggressive tumors, and there is currently no effective targeted therapy owing to the lack of specific mutation targets. Compared with lung adenocarcinoma (ADCs), lung SCCs reportedly utilized higher levels of glucose metabolism to meet the anabolic and catabolic needs required to sustain rapid tumor growth. Hexokinase 2 (HK2) is an enzyme that catalyzes the rate-limit and first committed step in glucose metabolism. Here, we investigated the expression and effect of HK2 in lung SCCs. We found a significantly higher HK2 expression in lung SCCs, but not lung ADC or normal tissues. HK2 depletion or inhibition decreased the glycolysis and tumor growth via activating AMPK signaling pathway, which downregulated mTORC1 activity. Furthermore, we found an increased oxygen respiration rate compensating for HK2 depletion. Thus, metformin treatment showed combinatorial therapeutic value, which resulted in greater induction of lung SCC apoptosis in vitro and in vivo. Our study suggests that HK2 depletion in combination with metformin might be a novel effective strategy for lung SCCs therapy.

PTGES/PGE2 signaling links immunosuppression and lung metastasis in Gprc5a-knockout mouse model.

Chronic inflammation has been linked to promotion of tumorigenesis and metastasis in lung. However, due to lack of a relevant animal model for characterization, the underlying mechanism remains elusive. Lung tumor suppressor gene Gprc5a-knockout (ko) mice are susceptible to lung inflammation, tumorigenesis and metastasis, which resembles the pathological features in human patients. Here, we showed that PTGES/PGE2 signaling was highly associated with lung tumorigenesis and metastasis in Gprc5a-ko mice. Interestingly, Ptges-knockout in mouse lung tumor cells, although reduced their stemness and EMT-like features, still formed tumors and lung metastasis in immune-deficient nude mice, but not in immune-competent mice. This suggests that the major role of PTGES/PGE2 signaling in tumorigenicity and lung metastasis is through immunosuppression. Mechanistically, PTGES/PGE2 signaling intrinsically endows tumor cells resistant to T-cell cytotoxicity, and induces cytokines extrinsically for MDSC recruitment, which is crucial for suppression of T-cell immunity. Importantly, targeting PGE2 signaling in Gprc5a-ko mice by PTGES inhibitor suppressed MDSC recruitment, restored T cells, and significantly repressed lung metastasis. Thus, PTGES/PGE2 signaling links immunosuppression and metastasis in an inflammatory lung microenvironment of Gprc5a-ko mouse model.

IL6/STAT3 Signaling Orchestrates Premetastatic Niche Formation and Immunosuppressive Traits in Lung.

Cancer cells that succeed in forming metastasis need to be reprogrammed to evade immune surveillance and survive in a new microenvironment. This is facilitated by metastatic niches that are either postformed through reciprocal signaling between tumor cells and local stromal cells or preformed as premetastatic niches before tumor cell arrival. IL6/STAT3 signaling is aberrantly activated in lung tumorigenesis and metastasis, however, the roles and mechanisms of action of IL6 remain controversial. Here, we showed that blockade of intrinsic STAT3 signaling in lung tumor cells suppressed lung metastasis in immune-competent syngeneic mice, but not in immune-deficient nude mice. Consistently, repression of STAT3 signaling in tumor cells made them susceptible to T-cell-mediated cytotoxicity. Thus, STAT3-mediated immunosuppression is crucial for metastasis. Noticeably, lung metastasis was greatly increased in Gprc5a-knockout (ko; 5a -/-) mice compared with wild-type mice, which correlated with upregulated IL6 in the tumor microenvironment. Depletion of IL6 via combined deletion of Il6 and Gprc5a genes almost completely eliminated lung metastasis in Gprc5a-ko/Il6-ko (5a -/-;Il6 -/-) mice. Mechanistically, dysregulated IL6 reprogrammed the STAT3 pathway in metastatic tumor cells, and induced recruitment of myeloid-derived suppressor cells and polarized macrophages to evade host immunity. Consistently, IHC staining showed that activated STAT3 correlated with repressed infiltration of CD8+ T cells in non-small cell lung cancer. Therefore, IL6/STAT3 signaling is crucial for orchestrating premetastatic niche formation and immunosuppression in lung.Significance: IL6 plays important roles not only in cell autonomous propensity for metastasis, but also in establishing the metastatic niche.

Stabilization of PTGES by deubiquitinase USP9X promotes metastatic features of lung cancer via PGE2 signaling.

Early metastasis and local recurrence are the major causes of mortality and poor prognosis of non-small cell lung cancer (NSCLC). However, the underlying mechanisms of these processes are poorly understood. In this study, we aimed to investigate the roles of the PTGES/PGE2 pathway in lung cancer progression. We found that prostaglandin E synthase (PTGES), a key enzyme for PGE2 synthesis in the arachidonic acid pathway, was highly dysregulated in NSCLC. Dysregulated PTGES was essential for the promotion of tumor migration and metastasis of NSCLC cells. Knockdown of PTGES in lung cancer cells resulted in suppressed cell migration, which was reversed by exogenous PGE2. Consistent with this, PTGES knockdown also reduced the expression of CSC markers, tumor sphere formation, colony forming activity, tumorigenicity, and lung metastasis in vivo. Dysregulated PTGES is mainly attributed to protein stabilization by USP9X, a deubiquitination enzyme. USP9X physically interacted with PTGES and prevented it from proteasome-directed degradation via deubiquitination. Consistent with this, USP9X expression was highly correlated with PTGES expression in NSCLC tumor tissues. Taken together, our results show that the upregulated USP9X-PTGES-PGE2 axis contributes significantly to the metastatic features of NSCLC.

ALDH2 Repression Promotes Lung Tumor Progression via Accumulated Acetaldehyde and DNA Damage.

The major role of aldehyde dehydrogenase 2 family (ALDH2) is to detoxify acetaldehyde (ACE) to non-toxic acetic acid. Many evidences suggest that ALDH2 dysfunction contributes to a variety of human diseases including cancer. However, the biological function and molecular mechanism of ALDH2 in tumor progression remain elusive. In this study, we found that ALDH2 repression was associated with poor prognosis in lung adenocarcinoma. Overexpression of ALDH2 inhibited malignant features of lung adenocarcinoma cells, such as proliferation, stemness and migration, whereas ALDH2 knockdown increased these features. Mechanistically, ALDH2 repression led to accumulation of ACE; whereas ACE enhanced the migration features of lung adenocarcinoma cells, which was associated with increased DNA damage. Importantly, accumulated ACE and increased DNA damage were identified in Aldh2-knockout (KO) mouse lung tissues in vivo. Consistent with this concept, treatment of lung adenocarcinoma cells with ALDH2 agonist Alda-1 suppressed the proliferation, stemness and migration features of lung adenocarcinoma cells. Thus, activating ALDH2, such as via its agonist, may provide a novel strategy for treatment of lung cancer.

p53 suppression is essential for oncogenic SPAG5 upregulation in lung adenocarcinoma.

Aberrant expression of sperm-associated antigen 5 (SPAG5) is implicated to play oncogenic roles in several types of cancers. However, the functions of SPAG5 in lung adenocarcinoma remain unclear. In this study, we investigated the role of SPAG5 in lung adenocarcinoma. We found that SPAG5 was upregulated in most of the lung adenocarcinoma cell lines as compared to normal lung epithelial cells. SPAG5 knockdown suppressed proliferation, colony forming, and migration of lung adenocarcinoma A549 cells in vitro and inhibited tumor growth in vivo. These suggest that upregulated SPAG5 promotes lung tumor progression. Importantly, treatment with MDM2 inhibitor, Nutlin-3a, restored p53 and p21 expression and suppressed SPAG5 expression in wild-type p53 lung adenocarcinoma cells, A549 and H460, but not in p53-null lung cancer cells, H1299. This suggests that the p53 signal pathway is essential for SPAG5 suppression. In addition, knocking-down p53 or p21 in A549 and H460 cells attenuated Nutlin-3a-induced repression of SPAG5, which further supports that the p53-p21 axis is required for SPAG5 repression. Thus, SPAG5 can serve as a prognostic marker, and therapeutic strategy targeting the p53-p21-SPAG5 axis may have important clinical implications.

Iron-dependent CDK1 activity promotes lung carcinogenesis via activation of the GP130/STAT3 signaling pathway.

Iron dysregulation is associated with several diseases, including lung cancer, but the underlying mechanism is yet unknown. Iron directly binds CDK1, which is upregulated in several cancers, thereby promoting JAK1 phosphorylation and activation of STAT3 signaling to promote colorectal carcinogenesis. This study aimed to investigate the role of iron/CDK1/STAT3 signaling in lung carcinogenesis. We found that iron-dependent CDK1 activity upregulated IL-6 receptor subunit GP130 post-transcriptionally via phosphorylation of 4E-BP1, which is critical for activation of JAK/STAT3 signaling. CDK1 and STAT3 are essential for iron-mediated colony formation in lung cancer cell lines. CDK1 knockdown and iron chelator DFO decreased tumorigenicity and GP130/STAT3 signaling in vivo. Moreover, CDK1/GP130/STAT3 signaling were elevated in lung cancer tissues compared with adjacent normal lung tissues. Altogether, the present results suggest that CDK1 inhibition and iron deprivation are potential strategies to target GP130/STAT3 signaling to suppress lung cancer.

Gprc5a depletion enhances the risk of smoking-induced lung tumorigenesis and mortality.

AIMS: Lung cancer remains the leading cause of cancer incidence and mortality. Although cigarette smoke is regarded as a high risk factor for lung tumor initiation, the role of the lung tumor suppressor GPRC5A in smoking-induced lung cancer is unclear. MAIN METHODS: We obtained two lung cancer cohorts from the TCGA and GEO databases. Bioinformatics analysis showed differential gene expression in the cohorts. Quantitative real-time PCR, Western Blot and Gprc5a-/- mice uncovered the relationship between cigarette smoke and lung cancer in the GPRC5A deletion system in vitro and in vivo. KEY FINDINGS: Bioinformatics analysis showed that the smoking lung cancer patients with low expression of GPRC5A had poor overall survival compared to the patients with high GPRC5A expression. Further analysis revealed that cancer-related stemness pathways such as the Hippo signaling pathway were induced in smoking patients with low GPRC5A expression. Additionally, we detected enriched expression of WNT5A and DLX5 in normal human lung epithelial 16HBE cells and human lung cancer H1299 cells in vitro. A relationship between cigarette smoke extract (NNK) and lung tumor initiation was observed in Gprc5a-/- mice. SIGNIFICANCE: The lung tumor suppressor gene GPRC5A played a protective role in cigarette smoke-induced lung tumor initiation, providing a target for the prevention of lung cancer development and monitoring of prognosis.

Dysregulated ENPP1 increases the malignancy of human lung cancer by inducing epithelial-mesenchymal transition phenotypes and stem cell features.

Induction of cancer stem cell (CSC) characters and epithelial mesenchymal transition (EMT) features are crucial in tumor initiation, progression and metastasis. However, underlying mechanisms remain incompletely understood. Here, we showed that ENPP1 plays an important role in inducing and maintaining EMT phenotypes and CSC features in lung cancer. ENPP1 is upregulated in lung cancer cells. ENPP1-knockdown in lung cancer HCC827 cells and A549 cells resulted in suppressed colonogenic formation, anchorage-independent growth in vitro, and tumorigenicity in vivo. ENPP1-knockdown also reduced expression of CSC makers, including ABCG2, SOX2, NANOG, and CD44. Moreover, ENPP1-knockdown reversed TGFß-induced EMT phenotypes, including cell migration, E-cadherin repression and vimentin induction. Finally, upregulated ENPP1 was identified in majority of human lung tumor tissues compared to adjacent normal lung tissues. Taken together, our study demonstrates that dysregulated ENPP1 contributes to increased malignancy of human lung cancer by inducing CSC-features, and EMT-like phenotypes.

GPRC5A deficiency leads to dysregulated MDM2 via activated EGFR signaling for lung tumor development.

GPRC5A, a retinoic acid induced gene, is preferentially expressed in lung tissue. Gprc5a gene deletion leads to spontaneous lung tumor development. However, the mechanism of Gprc5a-mediated lung tumor suppression is not fully understood. Here we showed that MDM2, a p53-negative regulator, was dysregulated in Gprc5a-knockout (ko) mouse tracheal epithelial cells (KO-MTEC) compared to wild type ones. Targeting MDM2 in 1601-a Gprc5a-ko mouse derived lung tumor cell line-and A549-human lung cancer cells, by MDM2 inhibitor Nutlin-3a or small hairpin RNA (sh-RNA)-restored p53 signaling pathway, reduced cancer stem cell markers, and inhibited tumorigenicity. This suggests that dysregulated MDM2 pathway is essential for the oncogenic activities of these cells. MDM2 was found to be stabilized mainly by activated EGFR signaling as targeting EGFR by Erlotinib or sh-RNA repressed MDM2 in a transcription-independent manner. Importantly, overexpression of MDM2 and reduced GPRC5A expression at both protein and mRNA levels were frequently found in clinical human lung cancer tissues. Taken together, GPRC5A deficiency contributes to dysregulated MDM2 via activated EGFR signaling, which promotes lung tumor development.