Putative Tumor Suppressor Research Articles

Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells

Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR-Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.

Genome-wide estrogen receptor β chromatin binding in human colon cancer cells reveals its tumor suppressor activity.

Colorectal cancer (CRC) is the third leading cause of cancer death in the western world. In women, menopausal hormone therapy has been shown to reduce CRC incidence by 20%. Studies demonstrate that estrogen activating estrogen receptor beta (ERβ) protects against CRC. ERβ is a nuclear receptor that regulates gene expression through interactions with the chromatin. This molecular mechanism is, however, not well characterized in colon. Here, we present for the first time, the cistrome of ERβ in different colon cancer cell lines. We use cell lines engineered to express ERβ, optimize and validate an ERβ antibody for chromatin-immunoprecipitation (ChIP), and perform ChIP-Seq. We identify key binding motifs, including ERE, AP-1, and TCF sites, and we determine enrichment of binding to cis-regulatory chromatin sites of genes involved in tumor development, cell migration, cell adhesion, apoptosis, and Wnt signaling pathways. We compare the corresponding cistromes of colon and breast cancer and find that they are conserved for about a third of genes, including GREB1, but that ERβ tethering to TCF and KLF family motifs is characteristic for colon. We exemplify upregulation of putative CRC tumor suppressor gene CST5 where ERβ in colon cells binds to cis-regulatory regions nearby (-351 bp) the transcriptional start site. Our work provides a foundation for understanding the mechanism of action of ERβ in CRC prevention.

DNA Methylation of Cannabinoid Receptor Interacting Protein 1 Promotes Pathogenesis of Intrahepatic Cholangiocarcinoma Through Suppressing Parkin-Dependent Pyruvate Kinase M2 Ubiquitination.

Methylation landscape is important for maintaining the silence of cannabinoid receptor-interacting protein 1 (CNRIP1) in some tumors. However, the role of CNRIP1 in intrahepatic cholangiocarcinoma (ICC) remains poorly defined. In our study, we showed that CNRIP1 was down-regulated in ICC tissues, and low expression of CNRIP1 was significantly associated with poor prognosis of patients with ICC in 3-year overall survival and tumor-free survival. Investigating the genomic DNA methylation profile, we disclosed a CpG island site named CNRIP1 MS-2 (CNRIP1 methylation site-2) that contributes to the down-regulation of CNRIP1. In addition, the methylation level of CNRIP1 MS-2 was correlated to the pathological grade, metastasis, and tumor-node-metastasis classification in ICC. Notably, we observed that CNRIP1 suppressed tumor cell migration, invasion, and proliferation by inhibiting the activity of pyruvate kinase M2 (PKM2). Sustained overexpression of CNRIP1 suppressed the in vivo tumor growth in a mouse xenograft model. It was also found that CNRIP1 overexpression activated Parkin (an E3 ubiquitin ligase), which resulted in the protein degradation of PKM2 in ICC cells. We identified that CNRIP1 acted as a putative tumor suppressor in ICC, which suggested that CNRIP1 could be a candidate biomarker for predicting tumor recurrence in patients with ICC. Furthermore, these findings highlight a potential therapeutic approach in targeting the CNRIP1/Parkin/PKM2 pathway for the treatment of ICC.

High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer.

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.

CSK-homologous kinase (CHK/MATK) is a potential colorectal cancer tumour suppressor gene epigenetically silenced by promoter methylation.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

The noncoding RNAs SNORD50A and SNORD50B-mediated TRIM21-GMPS interaction promotes the growth of p53 wild-type breast cancers by degrading p53

Small nucleolar RNA SNORD50A and SNORD50B (SNORD50A/B) has been reported to be recurrently deleted and function as a putative tumor suppressor in different types of cancer by binding to and suppressing the activity of the KRAS oncoproteins. Its deletion correlates with poorer patient survival. However, in this study, we surprisingly found that SNORD50A/B loss predicted a better survival in breast cancer patients carrying wild-type p53. Functional studies showed that SNORD50A/B deletion strongly inhibited the proliferation, migration, invasion and tumorigenic potential, and induced cell cycle arrest and apoptosis in p53 wild-type breast cancer cells, while exerted the opposite effects in p53 mutated breast cancer cells. This was also supported by ectopically expressing SNORD50A/B in both p53 wild-type and mutated breast cancer cells. Mechanistically, SNORD50A/B clearly enhances the interaction between E3 ubiquitin ligase TRIM21 and its substrate GMPS by forming a complex among them, thereby promoting GMPS ubiquitination and its subsequent cytoplasmic sequestration. SNORD50A/B deletion in p53 wild-type breast cancer cells will release GMPS and induce the translocation of GMPS into the nucleus, where GMPS can recruit USP7 and form a complex with p53, thereby decreasing p53 ubiquitination, stabilizing p53 proteins, and inhibiting malignant phenotypes of cancer cells. Altogether, the present study first reports that SNORD50A/B plays an oncogenic role in p53 wild-type breast cancers by mediating TRIM21-GMPS interaction.

Molecular Genetic and Genomic Alterations in Cushing's Syndrome and Primary Aldosteronism.

The genetic alterations that cause the development of glucocorticoid and/or mineralocorticoid producing benign adrenocortical tumors and hyperplasias have largely been elucidated over the past two decades through advances in genomics. In benign aldosterone-producing adrenocortical tumors and hyperplasias, alteration of intracellular calcium signaling has been found to be significant in aldosterone hypersecretion, with causative defects including those in KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. In benign cortisol-producing adrenocortical tumors and hyperplasias abnormal cyclic adenosine monophosphate-protein kinase A signaling has been found to play a central role in tumorigenesis, with pathogenic variants in GNAS, PRKAR1A, PRKACA, PRKACB, PDE11A, and PDE8B being implicated. The role of this signaling pathway in the development of Cushing’s syndrome and adrenocortical tumors was initially discovered through the study of the underlying genetic defects causing the rare multiple endocrine neoplasia syndromes McCune-Albright syndrome and Carney complex with subsequent identification of defects in genes affecting the cyclic adenosine monophosphate-protein kinase A pathway in sporadic tumors. Additionally, germline pathogenic variants in ARMC5, a putative tumor suppressor, were found to be a cause of cortisol-producing primary bilateral macronodular adrenal hyperplasia. This review describes the genetic causes of benign cortisol- and aldosterone-producing adrenocortical tumors.

Genetic Determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo.

In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2-the strongest drivers of growth in a KRAS-driven model-reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment. SIGNIFICANCE: By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease.This article is highlighted in the In This Issue feature, p. 1601.

Mast Cell-Derived SAMD14 Is a Novel Regulator of the Human Prostate Tumor Microenvironment.

Mast cells (MCs) are important cellular components of the tumor microenvironment and are significantly associated with poor patient outcomes in prostate cancer and other solid cancers. The promotion of tumor progression partly involves heterotypic interactions between MCs and cancer-associated fibroblasts (CAFs), which combine to potentiate a pro-tumor extracellular matrix and promote epithelial cell invasion and migration. Thus far, the interactions between MCs and CAFs remain poorly understood. To identify molecular changes that may alter resident MC function in the prostate tumor microenvironment, we profiled the transcriptome of human prostate MCs isolated from patient-matched non-tumor and tumor-associated regions of fresh radical prostatectomy tissue. Transcriptomic profiling revealed a distinct gene expression profile of MCs isolated from prostate tumor regions, including the downregulation of SAMD14, a putative tumor suppressor gene. Proteomic profiling revealed that overexpression of SAMD14 in HMC-1 altered the secretion of proteins associated with immune regulation and extracellular matrix processes. To assess MC biological function within a model of the prostate tumor microenvironment, HMC-1-SAMD14+ conditioned media was added to co-cultures of primary prostatic CAFs and prostate epithelium. HMC-1-SAMD14+ secretions were shown to reduce the deposition and alignment of matrix produced by CAFs and suppress pro-tumorigenic prostate epithelial morphology. Overall, our data present the first profile of human MCs derived from prostate cancer patient specimens and identifies MC-derived SAMD14 as an important mediator of MC phenotype and function within the prostate tumor microenvironment.

Targeting the Aryl Hydrocarbon Receptor Signaling Pathway in Breast Cancer Development.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.

LRP1B mutations are associated with favorable outcomes to immune checkpoint inhibitors across multiple cancer types

BackgroundLow-density lipoprotein receptor-related protein 1b (encoded by LRP1B) is a putative tumor suppressor, and preliminary evidence suggests LRP1B-mutated cancers may have improved outcomes with immune checkpoint inhibitors (ICI).MethodsWe conducted a...

A Functional Taxonomy of Tumor Suppression in Oncogenic KRAS-Driven Lung Cancer.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601.

Analysis of the P53N a Novel Protein Encoded on Chromosome 22q12.1-12.3 in Glioblastomas and Ependymomas Specimens

The P53N gene maps precisely to human chromosome sub-band 22q12.1-12.3, a region where loss of heterozygosity has been reported in 30% of astrocytic tumors and associated with progression to anaplasia. Moreover, a putative tumor suppressor gene has been indicated on 22q11 region involved in pathogenesis of ependymal tumors. Our objectives to examine the expression level of novel membrane-associated protein (termed P53N) encoded by a novel human gene on chromosome 22q12.1-12.3 in glioblastomas and ependymomas. Serial analysis of gene expression (SAGE) and immunofluorescence analysis of the P53N in the brain tumor tissues were performed. Our analysis revealed that there was high expression of the P53N mRNA in brain ependymoma and brain well-differentiated astrocytoma libraries. The P53N protein. P53N protein contains a high mobility group (HMG) domain at amino acid positions 301 to 360 expressed highly in glioblastoma and ependymoma specimens. Anti-P53N carboxyl-terminal peptide antibody localized the P53N protein to the cytoplasmic membranes of protoplasmic astrocytes in the glioblastoma and ependymoma specimens. These results are in good agreement with the SAGE analysis and the predicted transmembrane topology for the P53N protein and support a possible transmembrane model in which the P53N contains a predicted transmembrane region with its amino terminus localized to the inside of the cytoplasmic membrane.

Update on primary bilateral macronodular adrenal hyperplasia (PBMAH).

Primary bilateral macronodular adrenal hyperplasia (PBMAH), characterized by bilateral benign adrenal macronodules (>1 cm) potentially responsible for variable levels of cortisol excess, is a rare and heterogeneous disease. However, its frequency increases due to incidentally diagnosed cases on abdominal imaging carried out for reasons other than suspected adrenal disease. Mostly isolated, it can also be associated with dominantly inherited genetic conditions in rare cases. Considering the bilateral nature of adrenal involvement and the description of familial cases, the search of a genetic predisposition has led to the identification of germline heterozygous inactivating mutations of the putative tumor suppressor gene ARMC5, causing around 25% of the apparent sporadic cases. Rigorous biochemical and imaging assessment are key elements in the management of this challenging disease in terms of diagnosis. Treatment is reserved for symptomatic patients with overt or subclinical Cushing syndrome, and was historically based on bilateral adrenalectomy, which nowadays tends to be replaced by unilateral adrenalectomy or lifelong treatment with cortisol synthesis inhibitors.

Cellular Expression and Subcellular Localization of Wwox Protein During Testicular Development and Spermatogenesis in Rats.

A well-known putative tumor suppressor WW domain-containing oxidoreductase (Wwox) is highly expressed in hormonally regulated tissues and is considered important for the normal development and function of reproductive organs. In this study, we investigated the cellular and subcellular localization of Wwox in normal testes during postnatal days 0-70 using Western blotting and immunohistochemistry. Wwox is expressed in testes at all ages. Immunohistochemistry showed that fetal-type and adult-type Leydig cells, immature and mature Sertoli cells, and germ cells (from gonocytes to step 17 spermatids) expressed Wwox except peritubular myoid cells, step 18-19 spermatids, and mature sperm. Wwox localized diffusely in the cytoplasm with focal intense signals in all testicular cells. These signals gradually condensed in germ cells with their differentiation and colocalized with giantin for cis-Golgi marker and partially with golgin-97 for trans-Golgi marker. Biochemically, Wwox was detected in isolated Golgi-enriched fractions. But Wwox was undetectable in the nucleus. This subcellular localization pattern of Wwox was also confirmed in single-cell suspension. These findings indicate that Wwox is functional in most cell types of testis and might locate into Golgi apparatus via interaction with Golgi proteins. These unique localizations might be related to the function of Wwox in testicular development and spermatogenesis.

Abstract PO061: Prognostic significance of S100A2 expression in patients with resected lung adenocarcinoma

Abstract Background: Lung cancer is the leading cause of cancer death worldwide. The S100 protein family has been reported to regulate inflammatory responses, cell cycle progression, differentiation, and play an important role in initiation and progression of a variety of human cancers. Overexpression of S100A2 has been reported in a variety of cancer types. S100A2 has been reported to be a putative tumor suppressor in the early stages of carcinogenesis in lung cancer. However, other reports showed that S100A2 overexpression was significantly associated with poor survival and increased metastasis in patients with resected lung cancer. Due to the discrepancies in the direction of prognosis in the literature, the prognostic value of S100A2 in lung cancer needs further demonstration. Methods: The clinicopathological characteristics of 85 patients with lung adenocarcinoma at Taipei Veterans General Hospital were retrospectively reviewed. Expression of S100A2 was investigated using immunohistochemistry. Their prognostic significance in probability of overall survival and disease-free survival was analyzed. Results: With a median follow-up time of 26.7 months (26.8 ± 6.3), the 2-year probability of overall survival rate was 94.7%. By immunohistochemistry, overexpression of S100A2 was identified in 17 (20.0%) of the 85 patients. S100A2 overexpression was not a significant prognostic factor for overall survival (P = 0.122). For disease-free survival, patients with stage I disease have significantly better disease-free survival than those with stage II or III disease (P &amp;lt; 0.001) in univariate analysis. Patients with S100A2 overexpression have a significantly worse disease-free survival (P = 0.028). In multivariate analysis, S100A2 overexpression was still a significant prognostic factor for worse disease-free survival (P = 0.019). Stage I disease was also a significant prognostic factor for better disease-free survival (P = 0.006). Conclusions: Our study showed that S100A2 overexpression was a significant prognostic factor for worse disease-free survival in patients with resected lung adenocarcinoma. Citation Format: Jung-Jyh Hung. Prognostic significance of S100A2 expression in patients with resected lung adenocarcinoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO061.

CLIP4 Shows Putative Tumor Suppressor Characteristics in Breast Cancer: An Integrated Analysis.

Background: CAP-Gly domain containing linker protein family member 4 (CLIP4) plays an important role in cancers. However, its expression, prognostic value, and biological effect in breast cancer remain unclear. Methods: Data on patients diagnosed with breast cancer were retrieved from the TCGA-BRCA and other public omics databases. The expression profile of CLIP4 was analyzed using Oncomine, bc-GenExMiner, and TCGA. The prognostic value of CLIP4 was determined by Kaplan-Meier Plotter and Human Protein Atlas. Identification of genes co-expressed with CLIP4 and potential mechanism analyses were performed using UALCAN, STRING, Metascape, and GSEA. The epigenetic characteristics of CLIP4 were determined by DiseaseMeth and MEXPRESS. Results: CLIP4 was downregulated and its expression was negatively correlated with estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor type 2 (HER2) status, Nottingham prognostic index (NPI), and Scarff-Bloom-Richardson (SBR) grade in breast cancer, whereas it was positively linked to basal-like and triple negative breast cancer status. Ectopic expression of CLIP4 was related with poor prognosis. In the analysis of genes co-expressed with CLIP4, GSEA showed that the Hedgehog (Hh), JAK-STAT, ERBB, Wnt signaling pathway, cell adhesion molecules, and pathways in cancer were dissimilarly enriched in the CLIP4 expression high phenotype. Analysis of the genetics and epigenetics of CLIP4 indicated that its expression was negatively correlated with DNA methylation. Conclusion: Methylated CLIP4 may be a novel prognostic and therapeutic biomarker for breast cancer.

Loss of G-Protein Pathway Suppressor 2 Promotes Tumor Growth Through Activation of AKT Signaling.

G Protein Suppressor 2 (GPS2) is a multifunctional protein that exerts important roles in inflammation and metabolism in adipose, liver, and immune cells. GPS2 has recently been identified as a significantly mutated gene in breast cancer and other malignancies and proposed to work as a putative tumor suppressor. However, molecular mechanisms by which GPS2 prevents cancer development and/or progression are largely unknown. Here, we have profiled the phenotypic changes induced by GPS2 depletion in MDA-MB-231 triple negative breast cancer cells and investigated the underlying molecular mechanisms. We found that GPS2-deleted MDA-MB-231 cells exhibited increased proliferative, migratory, and invasive properties in vitro, and conferred greater tumor burden in vivo in an orthotopic xenograft mouse model. Transcriptomic, proteomic and phospho-proteomic profiling of GPS2-deleted MBA-MB-231 revealed a network of altered signals that relate to cell growth and PI3K/AKT signaling. Overlay of GPS2-regulated gene expression with MDA-MB-231 cells modified to express constitutively active AKT showed significant overlap, suggesting that sustained AKT activation is associated with loss of GPS2. Accordingly, we demonstrate that the pro-oncogenic phenotypes associated with GPS2 deletion are rescued by pharmacological inhibition of AKT with MK2206. Collectively, these observations confirm a tumor suppressor role for GPS2 and reveal that loss of GPS2 promotes breast cancer cell proliferation and tumor growth through uncontrolled activation of AKT signaling. Moreover, our study points to GPS2 as a potential biomarker for a subclass of breast cancers that would be responsive to PI3K-class inhibitor drugs.

Proteomic identification and validation of novel interactions of the putative tumor suppressor PRELP with membrane proteins including IGFI-R and p75NTR

Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP’s links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.

Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.