Landscape of tumoral ecosystem for enhanced anti-PD-1 immunotherapy by gut Akkermansia muciniphila.

Gut Akkermansia muciniphila (Akk) has been implicated in impacting immunotherapy or oncogenesis. This study aims to dissect the Akk-associated tumor immune ecosystem (TIME) by single-cell profiling coupled with T cell receptor (TCR) sequencing. We adopted mouse cancer models under anti-PD-1 immunotherapy, combined with oral administration of three forms of Akk, including live Akk, pasteurized Akk (Akk-past), or its membrane protein Amuc_1100 (Amuc). We show that live Akk is most effective in activation of CD8 T cells by rescuing the exhausted type into cytotoxic subpopulations. Remarkably, only live Akk activates MHC-II-pDC pathways, downregulates CXCL3 in Bgn(+)Dcn(+) cancer-associated fibroblasts (CAFs), blunts crosstalk between Bgn(+)Dcn(+) CAFs and PD-L1(+) neutrophils by a CXCL3-PD-L1 axis, and further suppresses the crosstalk between PD-L1(+) neutrophils and CD8 T cells, leading to the rescue of exhausted CD8 T cells. Together, this comprehensive picture of the tumor ecosystem provides deeper insights into immune mechanisms associated with gut Akk-dependent anti-PD-1 immunotherapy.

COVID-19 vaccination is associated with enhanced efficacy of anti-PD-(L)1 immunotherapy in advanced NSCLC patients: a real-world study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) vaccine has played a major role in ending the pandemic. However, little is known about the influence of COVID-19 vaccine on the efficacy of immunotherapy in patients with non-small cell lung cancer (NSCLC). OBJECTIVES: The goal of this study is to explore whether COVID-19 vaccine impacts the efficacy of immune checkpoint inhibitors (ICIs) in NSCLC patients. METHODS: We retrospectively analyzed the survival data of ICI-treated 104 patients with stage III-IV NSCLC, who either received COVID-19 vaccination (n = 25) or no vaccination (n = 79). The potential risk factors, in particular roles of COVID-19 vaccination in the efficacy of ICIs in these patients, were evaluated. RESULTS: Our results showed significantly improved ORR (28.0% vs. 11.39%, p = 0.05) and DCR (88.0% vs. 54.43%, p = 0.005) in the COVID-19 vaccinated group compared with the non-vaccinated group. Regarding the long-term survival benefits, COVID-19 vaccine showed profound influence both on the PFS (HR = 0.16, p = 0.021) and OS (HR = 0.168, p = 0.019) in patients with NSCLC under ICIs treatment. The PFS (p < 0.001) or OS (p < 0.001) was significantly improved in the COVID-19 vaccinated group, compared with the non-vaccinated group. Moreover, CD4 T cell (p = 0.047) level was higher in the COVID-19 vaccinated group than in the non-vaccinated group. CONCLUSIONS: COVID-19 vaccination enhances anti-PD-1 immunotherapy efficacy in patients with stage III-IV NSCLC, suggesting that COVID-19 vaccination may provide additional benefit to NSCLC patients.

N6-methyladenosine modified LINC00901 promotes pancreatic cancer progression through IGF2BP2/MYC axis.

Accumulating evidence indicates that RNA methylation at N6-methyladenosine (m6A) plays an important regulatory role in gene expression and aberrant mRNA m6A modification is often associated with a variety of cancers. However, little is known whether and how m6A-modification impacts long non-coding RNA (lncRNA) and lncRNA-mediated tumorigenesis, particularly in pancreatic ductal adenocarcinoma (PDAC). In the present study, we report that a previously uncharacterized lncRNA, LINC00901, promotes pancreatic cancer cell growth and invasion and moreover, LINC00901 is subject to m6A modification which regulates its expression. In this regard, YTHDF1 serves as a reader for the m6A modified LINC00901 and downregulates the LINC00901 level. Notably, two conserved m6A sites in LINC00901 are critical to the recognition of LINC00901 by YTHDF1. Finally, RNA sequencing (RNA-seq) and gene function analysis revealed that LINC00901 positively regulates MYC through upregulation of IGF2BP2, a known RNA binding protein that can enhance MYC mRNA stability. Together, our results suggest that there is a LINC00901-IGF2BP2-MYC axis through which LINC00901 promotes PDAC progression in an m6A dependent manner.

Circulating eosinophils associated with responsiveness to COVID-19 vaccine and the disease severity in patients with SARS-CoV-2 omicron variant infection.

OBJECTIVE: This study aimed to investigate the longitudinal circulating eosinophil (EOS) data impacted by the COVID-19 vaccine, the predictive role of circulating EOS in the disease severity, and its association with T cell immunity in patients with SARS-CoV-2 Omicron BA.2 variant infection in Shanghai, China. METHODS: We collected a cohort of 1,157 patients infected with SARS-CoV-2 Omicron/BA.2 variant in Shanghai, China. These patients were diagnosed or admitted between Feb 20, 2022, and May 10, 2022, and were classified as asymptomatic (n = 705), mild (n = 286) and severe (n = 166) groups. We compiled and analyzed data of patients' clinical demographic characteristics, laboratory findings, and clinical outcomes. RESULTS: COVID-19 vaccine reduced the incidence of severe cases. Severe patients were shown to have declined peripheral blood EOS. Both the 2 doses and 3 doses of inactivated COVID-19 vaccines promoted the circulating EOS levels. In particular, the 3rd booster shot of inactivated COVID-19 vaccine was shown to have a sustained promoting effect on circulating EOS. Univariate analysis showed that there was a significant difference in age, underlying comorbidities, EOS, lymphocytes, CRP, CD4, and CD8 T cell counts between the mild and the severe patients. Multivariate logistic regression analysis and ROC curve analysis indicate that circulating EOS (AUC = 0.828, p = 0.025), the combination of EOS and CD4 T cell (AUC = 0.920, p = 0.017) can predict the risk of disease severity in patients with SARS-CoV-2 Omicron BA.2 variant infection. CONCLUSIONS: COVID-19 vaccine promotes circulating EOS and reduces the risk of severe illness, and particularly the 3rd booster dose of COVID-19 vaccine sustainedly promotes EOS. Circulating EOS, along with T cell immunity, may have a predictive value for the disease severity in SARS-CoV-2 Omicron infected patients.

Microbiome and spatially resolved metabolomics analysis reveal the anticancer role of gut Akkermansia muciniphila by crosstalk with intratumoral microbiota and reprogramming tumoral metabolism in mice.

Although gut microbiota has been linked to cancer, little is known about the crosstalk between gut- and intratumoral-microbiomes. The goal of this study was to determine whether gut Akkermansia muciniphila (Akk) is involved in the regulation of intratumoral microbiome and metabolic contexture, leading to an anticancer effect on lung cancer. We evaluated the effects of gut endogenous or gavaged exogenous Akk on the tumorigenesis using the Lewis lung cancer mouse model. Feces, blood, and tumor tissue samples were collected for 16S rDNA sequencing. We then conducted spatially resolved metabolomics profiling to discover cancer metabolites in situ directly and to characterize the overall Akk-regulated metabolic features, followed by the correlation analysis of intratumoral bacteria with metabolic network. Our results showed that both endogenous and exogenous gavaged Akk significantly inhibited tumorigenesis. Moreover, we detected increased Akk abundance in blood circulation or tumor tissue by 16S rDNA sequencing in the Akk gavaged mice, compared with the control mice. Of great interest, gavaged Akk may migrate into tumor tissue and influence the composition of intratumoral microbiome. Spatially resolved metabolomics analysis revealed that the gut-derived Akk was able to regulate tumor metabolic pathways, from metabolites to enzymes. Finally, our study identified a significant correlation between the gut Akk-regulated intratumoral bacteria and metabolic network. Together, gut-derived Akk may migrate into blood circulation, and subsequently colonize into lung cancer tissue, which contributes to the suppression of tumorigenesis by influencing tumoral symbiotic microbiome and reprogramming tumoral metabolism, although more studies are needed.

Exosomal miR-1304-3p promotes breast cancer progression in African Americans by activating cancer-associated adipocytes.

Breast cancer displays disparities in mortality between African Americans and Caucasian Americans. However, the exact molecular mechanisms remain elusive. Here, we identify miR-1304-3p as the most upregulated microRNA in African American patients. Importantly, its expression significantly correlates with poor progression-free survival in African American patients. Ectopic expression of miR-1304 promotes tumor progression in vivo. Exosomal miR-1304-3p activates cancer-associated adipocytes that release lipids and enhance cancer cell growth. Moreover, we identify the anti-adipogenic gene GATA2 as the target of miR-1304-3p. Notably, a single nucleotide polymorphism (SNP) located in the miR-1304 stem-loop region shows a significant difference in frequencies of the G allele between African and Caucasian American groups, which promotes the maturation of miR-1304-3p. Therefore, our results reveal a mechanism of the disparity in breast cancer progression and suggest a potential utility of miR-1304-3p and the associated SNP as biomarkers for predicting the outcome of African American patients.

LncRNA IPW inhibits growth of ductal carcinoma in situ by downregulating ID2 through miR-29c.

BACKGROUND: Ductal carcinoma in situ (DCIS) of breast is the noninvasive lesion that has propensity to progress to the malignant form. At present, it is still unknown which lesions can potentially progress to invasive forms. In this study, we aimed to identify key lncRNAs involved in DCIS growth. METHODS: We employ disease-related lncProfiler array to identify IPW in specimens of DCIS and matching control samples and validate the observations in three DCIS-non-tumorigenic cell lines. Further, we examine the mechanism of IPW action and the downstream signaling in in vitro and in vivo assays. Importantly, we screened a library containing 390 natural compounds to identify candidate compound selectively inhibiting IPW low DCIS cells. RESULTS: We identified lncRNA IPW as a novel tumor suppressor critical for inhibiting DCIS growth. Ectopic expression of IPW in DCIS cells strongly inhibited cell proliferation, colony formation and cell cycle progression while silencing IPW in primary breast cells promoted their growth. Additionally, orthotropic implantation of cells with ectopic expression of IPW exhibited decreased tumor growth in vivo. Mechanistically, IPW epigenetically enhanced miR-29c expression by promoting H3K4me3 enrichment in its promoter region. Furthermore, we identified that miR-29c negatively regulated a stemness promoting gene, ID2, and diminished self-renewal ability of DCIS cells. Importantly, we screened a library containing 390 natural compounds and identified toyocamycin as a compound that selectively inhibited the growth of DCIS with low expression of IPW, while it did not affect DCIS with high IPW expression. Toyocamycin also suppressed genes associated with self-renewal ability and inhibited DCIS growth in vivo. CONCLUSION: Our findings revealed a critical role of the IPW-miR-29c-ID2 axis in DCIS formation and suggested potential clinical use of toyocamycin for the treatment of DCIS.

Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

BACKGROUND: UCA1 is frequently upregulated in a variety of cancers, including CRC, and it can play an oncogenic role by various mechanisms. However, how UCA1 is regulated in cancer is largely unknown. In this study, we aimed to determine whether RNA methylation at N6-methyladenosine (m6A) can impact UCA1 expression in colorectal cancer (CRC). METHODS: qRT-PCR was performed to detect the level of UCA1 and IGF2BP2 in CRC samples. CRISPR/Cas9 was employed to knockout (KO) UCA1, METTL3 and WTAP in DLD-1 and HCT-116 cells, while rescue experiments were carried out to re-express METTL3 and WTAP in KO cells. Immunoprecipitation using m6A antibody was performed to determine the m6A modification of UCA1. In vivo pulldown assays using S1m tagging combined with site-direct mutagenesis was carried out to confirm the recognition of m6A-modified UCA1 by IGF2BP2. Cell viability was measured by MTT and colony formation assays. The expression of UCA1 and IGF2BP2 in TCGA CRC database was obtained from GEPIA ( http://gepia.cancer-pku.cn ). RESULTS: Our results revealed that IGF2BP2 serves as a reader for m6A modified UCA1 and that adenosine at 1038 of UCA1 is critical to the recognition by IGF2BP2. Importantly, we showed that m6A writers, METTL3 and WTAP positively regulate UCA1 expression. Mechanically, IGF2BP2 increases the stability of m6A-modified UCA1. Clinically, IGF2BP2 is upregulated in CRC tissues compared with normal tissues. CONCLUSION: These results suggest that m6A modification is an important factor contributing to upregulation of UCA1 in CRC tissues.

Lnc-DC promotes estrogen independent growth and tamoxifen resistance in breast cancer.

Selective estrogen receptor modulators (SERMs) such as tamoxifen have proven to be effective in the treatment of estrogen receptor (ER) positive breast cancer. However, a major obstacle for such endocrine therapy is estrogen independent growth, leading to resistance, and the underlying mechanism is not fully understood. The purpose of this study was to determine whether long non-coding RNAs (lncRNAs) are involved in regulation of estrogen independent growth and tamoxifen resistance in ER positive breast cancer. Using a CRISPR/Cas9-based SAM (synergistic activation mediator) library against a focus group of lncRNAs, we identify Lnc-DC as a candidate lncRNA. Further analysis suggests that Lnc-DC is able to reduce tamoxifen-induced apoptosis by upregulation of anti-apoptotic genes such as Bcl2 and Bcl-xL. Furthermore, Lnc-DC activates STAT3 by phosphorylation (pSTAT3Y705), and the activated STAT3 subsequently induces expression of cytokines which in turn activate STAT3, forming an autocrine loop. Clinically, upregulation of Lnc-DC is associated with poor prognosis. In particular, analysis of a tamoxifen-treated patient cohort indicates that Lnc-DC expression can predict the response to tamoxifen. Together, this study demonstrates a previously uncharacterized function of Lnc-DC/STAT3/cytokine axis in estrogen independent growth and tamoxifen resistance, and Lnc-DC may serve as a potential predictor for tamoxifen response.

Exosomal miR-19a and IBSP cooperate to induce osteolytic bone metastasis of estrogen receptor-positive breast cancer.

Bone metastasis is an incurable complication of breast cancer. In advanced stages, patients with estrogen-positive tumors experience a significantly higher incidence of bone metastasis (>87%) compared to estrogen-negative patients (<56%). To understand the mechanism of this bone-tropism of ER+ tumor, and to identify liquid biopsy biomarkers for patients with high risk of bone metastasis, the secreted extracellular vesicles and cytokines from bone-tropic breast cancer cells are examined in this study. Both exosomal miR-19a and Integrin-Binding Sialoprotein (IBSP) are found to be significantly upregulated and secreted from bone-tropic ER+ breast cancer cells, increasing their levels in the circulation of patients. IBSP is found to attract osteoclast cells and create an osteoclast-enriched environment in the bone, assisting the delivery of exosomal miR-19a to osteoclast to induce osteoclastogenesis. Our findings reveal a mechanism by which ER+ breast cancer cells create a microenvironment favorable for colonization in the bone. These two secreted factors can also serve as effective biomarkers for ER+ breast cancer to predict their risks of bone metastasis. Furthermore, our screening of a natural compound library identifies chlorogenic acid as a potent inhibitor for IBSP-receptor binding to suppress bone metastasis of ER+ tumor, suggesting its preventive use for bone recurrence in ER+ patients.

Epigenetic and Posttranscriptional Modulation of SOS1 Can Promote Breast Cancer Metastasis through Obesity-Activated c-Met Signaling in African-American Women.

Ethnicity is considered to be one of the major risk factors in certain subtypes of breast cancer. However, the mechanism of this racial disparity remains poorly understood. Here, we demonstrate that SOS1, a key regulator of Ras pathway, is highly expressed in African-American (AA) patients with breast cancer compared with Caucasian-American patients. Because of the higher obesity rate in AA women, increased levels of SOS1 facilitated signal transduction of the c-Met pathway, which was highly activated in AA patients with breast cancer via hepatocyte growth factor secreted from adipocytes. Elevated expression of SOS1 also enhanced cancer stemness through upregulation of PTTG1 and promoted M2 polarization of macrophages by CCL2 in metastatic sites. SOS1 was epigenetically regulated by a super-enhancer identified by H3K27ac in AA patients. Knockout of the super-enhancer by CRISPR in AA cell lines significantly reduced SOS1 expression. Furthermore, SOS1 was posttranscriptionally regulated by miR-483 whose expression is reduced in AA patients through histone trimethylation (H3K27me3) on its promoter. The natural compound, taxifolin, suppressed signaling transduction of SOS1 by blocking the interaction between SOS1 and Grb2, suggesting a potential utility of this compound as a therapeutic agent for AA patients with breast cancer. SIGNIFICANCE: These findings elucidate the signaling network of SOS1-mediated metastasis in African-American patients, from the epigenetic upregulation of SOS1 to the identification of taxifolin as a potential therapeutic strategy against SOS1-driven tumor progression.

Comprehensive Network Analysis Reveals Alternative Splicing-Related lncRNAs in Hepatocellular Carcinoma.

It is increasingly appreciated that long non-coding RNAs (lncRNAs) associated with alternative splicing (AS) could be involved in aggressive hepatocellular carcinoma. Although many recent studies show the alteration of RNA alternative splicing by deregulated lncRNAs in cancer, the extent to which and how lncRNAs impact alternative splicing at the genome scale remains largely elusive. We analyzed RNA-seq data obtained from 369 hepatocellular carcinomas (HCCs) and 160 normal liver tissues, quantified 198,619 isoform transcripts, and identified a total of 1,375 significant AS events in liver cancer. In order to predict novel AS-associated lncRNAs, we performed an integration of co-expression, protein-protein interaction (PPI) and epigenetic interaction networks that links lncRNA modulators (such as splicing factors, transcript factors, and miRNAs) along with their targeted AS genes in HCC. We developed a random walk-based multi-graphic (RWMG) model algorithm that prioritizes functional lncRNAs with their associated AS targets to computationally model the heterogeneous networks in HCC. RWMG shows a good performance evaluated by the ROC curve based on cross-validation and bootstrapping strategies. As a conclusion, our robust network-based framework has derived 31 AS-related lncRNAs that not only validates known cancer-associated cases MALAT1 and HOXA11-AS, but also reveals new players such as DNM1P35 and DLX6-AS1with potential functional implications. Survival analysis further provides insights into the clinical significance of identified lncRNAs.

Gut microbiota regulate tumor metastasis via circRNA/miRNA networks.

BACKGROUND: Increasing evidence indicates that gut microbiota plays an important role in cancer progression. However, the underlying mechanism remains largely unknown. Here, we report that broad-spectrum antibiotics (ABX) treatment leads to enhanced metastasis by the alteration of gut microbiome composition. METHODS: Cancer LLC and B16-F10 cell metastasis mouse models, and microarray/RNA sequencing analysis were used to reveal the regulatory functions of microbiota-mediated circular RNA (circRNA)/microRNA (miRNA) networks that may contribute to cancer metastasis. RESULTS: The specific pathogen-free (SPF) mice with ABX treatment demonstrated enhanced lung metastasis. Fecal microbiota transplantation (FMT) from SPF mice or Bifidobacterium into germ-free mice significantly suppressed lung metastasis. Mechanistically, gut microbiota impacts circRNA expression to regulate levels of corresponding miRNAs. Specifically, such modulations of gut microbiota inhibit mmu_circ_0000730 expression in an IL-11-dependent manner. Bioinformatics analysis combined with luciferase reporter assays revealed reciprocal repression between mmu_circ_0000730 and mmu-miR-466i-3p. We further showed that both mmu-miR-466i-3p and mmu-miR-466 f-3p suppresses a number of genes involved in epithelial-mesenchymal transition (EMT) and stemness of cancer stem cells such as SOX9. CONCLUSIONS: These results provide evidence of a previously unrecognized regulatory role of non-coding RNAs in microbiota-mediated cancer metastasis, and thus, the microbiome may serve as a therapeutic target.