Activation of the PI3K/AKT signaling pathway by ARNTL2 enhances cellular glycolysis and sensitizes pancreatic adenocarcinoma to erlotinib.

BACKGROUND: Pancreatic adenocarcinoma (PC) is an aggressive malignancy with limited treatment options. The poor prognosis primarily stems from late-stage diagnosis and when the disease has become therapeutically challenging. There is an urgent need to identify specific biomarkers for cancer subtyping and early detection to enhance both morbidity and mortality outcomes. The addition of the EGFR tyrosine kinase inhibitor (TKI), erlotinib, to gemcitabine chemotherapy for the first-line treatment of patients with advanced pancreatic cancer slightly improved outcomes. However, restricted clinical benefits may be linked to the absence of well-characterized criteria for stratification and dependable biomarkers for the prediction of treatment effectiveness. METHODS AND RESULTS: We examined the levels of various cancer hallmarks and identified glycolysis as the primary risk factor for overall survival in PC. Subsequently, we developed a glycolysis-related score (GRS) model to accurately distinguish PC patients with high GRS. Through in silico screening of 4398 compounds, we discovered that erlotinib had the strongest therapeutic benefits for high-GRS PC patients. Furthermore, we identified ARNTL2 as a novel prognostic biomarker and a predictive factor for erlotinib treatment responsiveness in patients with PC. Inhibition of ARNTL2 expression reduced the therapeutic efficacy, whereas increased expression of ARNTL2 improved PC cell sensitivity to erlotinib. Validation in vivo using patient-derived xenografts (PDX-PC) with varying ARNTL2 expression levels demonstrated that erlotinib monotherapy effectively halted tumor progression in PDX-PC models with high ARNTL2 expression. In contrast, PDX-PC models lacking ARNTL2 did not respond favorably to erlotinib treatment. Mechanistically, we demonstrated that the ARNTL2/E2F1 axis-mediated cellular glycolysis sensitizes PC cells to erlotinib treatment by activating the PI3K/AKT signaling pathway. CONCLUSIONS: Our investigations have identified ARNTL2 as a novel prognostic biomarker and predictive indicator of sensitivity. These results will help to identify erlotinib-responsive cases of PC and improve treatment outcomes. These findings contribute to the advancement of precision oncology, enabling more accurate and targeted therapeutic interventions.

Cuproptosis-related lncRNAs are correlated with tumour metabolism and immune microenvironment and predict prognosis in pancreatic cancer patients.

Cuproptosis is a novel cell death pathway, and the regulatory mechanism in pancreatic cancer (PC) is unclear. The authors aimed to figure out whether cuproptosis-related lncRNAs (CRLs) could predict prognosis in PC and the underlying mechanism. First, the prognostic model based on seven CRLs screened by the least absolute shrinkage and selection operator Cox analysis was constructed. Following this, the risk score was calculated for pancreatic cancer patients and divided patients into high and low-risk groups. In our prognostic model, PC patients with higher risk scores had poorer outcomes. Based on several prognostic features, a predictive nomogram was established. Furthermore, the functional enrichment analysis of differentially expressed genes between risk groups was performed, indicating that endocrine and metabolic pathways were potential regulatory pathways between risk groups. TP53, KRAS, CDKN2A, and SMAD4 were dominant mutated genes in the high-risk group and tumour mutational burden was positively correlated with the risk score. Finally, the tumour immune landscape indicated patients in the high-risk group were more immunosuppressive than that in the low-risk group, with lower infiltration of CD8+ T cells and higher M2 macrophages. Above all, CRLs can be applied to predict PC prognosis, which is closely correlated with the tumour metabolism and immune microenvironment.

Chemotherapeutic drugs-induced pyroptosis mediated by gasdermin E promotes the progression and chemoresistance of pancreatic cancer.

Pyroptosis is closely associated with cancer development; however, the role of pyroptosis in pancreatic ductal adenocarcinoma (PDAC), a fatal malignant tumour with a poor overall survival rate, remains elusive. Here, we explored the mechanism of chemotherapy-induced pyroptosis and elucidated the role of pyroptosis in mediating PDAC progression and chemoresistance. The results demonstrated first- and second-line chemotherapeutic drugs against PDAC, including gemcitabine, irinotecan, 5-fluorouracil, paclitaxel, and cisplatin, induced concurrent pyroptosis and apoptosis. During this process, gasdermin E (GSDME) was cleaved by activated caspase-3, which was accompanied by pro-apoptotic caspase-7/8 activation. GSDME knockdown switched pyroptosis to apoptosis, decreased invasion and migration, and enhanced the sensitivity of PDAC cells to chemotherapy in vitro and in vivo. GSDME was highly expressed in PDAC tissues and positively correlated with histological differentiation and vascular invasion. Furthermore, cells that survived pyroptosis promoted proliferation and invasion and impaired the chemosensitivity of PDAC cells, which was attenuated by the GSDME knockdown. Our findings demonstrated that chemotherapeutics against PDAC induce GSDME-dependent pyroptosis, and GSDME expression positively correlated with PDAC progression and chemoresistance. Targeting GSDME may be a novel approach to overcoming chemoresistance in PDAC.

PLA2G2A+ cancer-associated fibroblasts mediate pancreatic cancer immune escape via impeding antitumor immune response of CD8+ cytotoxic T cells.

Our previous research defined a novel metabolic cancer associated fibroblasts subset (meCAFs) enriched in loose-type pancreatic ductal adenocarcinoma (PDAC) and related to CD8+ T cells accumulation. Consistently, the abundance of meCAFs was associated with poor prognosis but better immunotherapy responses in PDAC patients. However, the metabolic characteristic of meCAFs and its cross-talk with CD8+ T cells remain to be elucidated. In this study, we identified PLA2G2A as a marker of meCAFs. In particular, the abundance of PLA2G2A+ meCAFs was positively related to the accumulation of total CD8+ T cells and negatively correlated with clinical outcomes of PDAC patients and infiltration of intratumoral CD8+ T cells. We demonstrated that PLA2G2A+ meCAFs substantially attenuated the antitumor ability of tumor infiltrating CD8+ T cells and facilitated tumor immune escape in PDAC. Mechanistically, PLA2G2A regulated the function of CD8+ T cells as a pivotal soluble mediator via MAPK/Erk and NF-κB signaling pathways. In conclusion, our study identified the unrecognized role of PLA2G2A+ meCAFs in promoting tumor immune escape by impeding the antitumor immune function of CD8+ T cells, and strongly suggested PLA2G2A as a promising biomarker and therapeutic target for immunotherapy in PDAC.

HDACs/mTOR inhibitor synergizes with pyrotinib in HER2-positive pancreatic cancer through degradation of mutant P53.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), as a highly lethal malignancy with high mortality, lacks of effective treatment. Canonical therapeutic targets in PDAC demand further verification among which HER2 receptor tyrosine kinase inhibitor pyrotinib as treatment targets has not be decided. METHODS: Anti-PDAC efficacy of pyrotinib was evaluated both in vitro and in vivo using both cell lines and patient-derived xenografts. By screening a large-scale library of 1453 compounds, we identified HDACs/mTOR inhibitor 1 as a promising candidate to synergize with pyrotinib. The combination therapy was evaluated in vitro and in vivo in multiple cell lines and animal models. Furthermore, RNA-seq analysis was performed to reveal the latent molecular mechanism of combination therapy. RESULTS: In our study, pyrotinib monotherapy was found to be inefficient to anti-PDAC which exhibited limited anti-proliferation effect in vitro and in vivo. Through therapy combined with HDACs/mTOR inhibitor 1, pyrotinib triggered intense apoptosis in PDAC both in cell lines and animal models. Mechanistic analyses revealed that mutant P53 degradation mediated by HDAC inhibition synergized with HER2 and mTOR inhibition. CONCLUSIONS: In conclusion, identification of HDACs/mTOR inhibitor as a synergistic inhibitor, provides a potent therapeutic strategy that targets HER2-positive pancreatic cancer.

A novel angiogenesis-based molecular signature related to prognosis and tumor immune interactions of pancreatic cancer.

Angiogenesis, a hallmark of cancer, is related to prognosis, tumor progression, and treatment response. Nevertheless, the correlation of angiogenesis-based molecular signature with clinical outcome and immune cell infiltration has not been thoroughly studied in pancreatic cancer. In this study, multiple bioinformatics methods were combined to evaluate prognosis, immune cell infiltration, and the alterations of angiogenesis-related genes (ARGs) in PC samples, and further establish a novel angiogenesis-related gene signature. Moreover, the protein and mRNA expression levels of four angiogenesis risk genes were determined by Human Protein Atlas (HPA) database and qPCR analysis, respectively. Here, we recognized two distinct angiogenesis subtypes and two gene subtypes, and revealed the critical roles of ARGs in the tumor immune microenvironment (TIME), clinical features, and prognosis. Consequently, we established an ARGs score to predict prognosis and therapeutic response of PC patients, and validated its robust predictive ability. Additionally, the ARGs score was markedly associated with clinical outcomes, tumor mutation burden (TMB), and chemotherapeutic drug sensitivity. In brief, our findings imply that the ARGs score is a robust prognostic indicator and may contribute to the development of effective individualized therapies for PC.

Association of aging-related genes with prognosis and immune infiltration in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is one of the deadliest malignancies. Aging is described as the degeneration of physiological function, which is complexly correlated with cancer. It is significant to explore the influences of aging-related genes (ARGs) on PAAD. Based on The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets, we used univariate Cox regression analysis and acquired eight differentially expressed ARGs with prognostic values. Two molecular subtypes were identified based on these ARGs to depict PAAD patients' overall survival (OS) and immune microenvironments preliminarily. Cluster 1 had a poor OS as well as a worse immune microenvironment. Through least absolute shrinkage and selection operator (LASSO) regression analysis, we constructed a seven-ARG risk signature based on the TCGA dataset and verified it in Gene Expression Omnibus (GEO) and International Cancer Genome Consortium (ICGC) to predict the prognoses, immune microenvironments, signal pathways, tumor mutations, and drug sensitivity of PAAD patients. The high-risk group possessed an unfavorable OS compared with that of the low-risk group. We also verified the independence and clinical availability of the risk signature by Cox regression analyses and the establishment of a nomogram, respectively. The higher risk score was associated with several clinical factors such as higher grade and advanced tumor stage as well as lower immunoscore and cluster 1. The negative associations of risk scores with immune, stroma, and estimate scores proved the terrible immune microenvironment in the high-risk group. Relationships between risk score and immune checkpoint gene expression as well as signal pathways provided several therapeutic targets. PAAD patients in the low-risk group possessed lower tumor mutations as well as a higher susceptibility to axitinib and vorinostat. The high-risk group bore a higher TMB and cisplatin and dasatinib may be better options. We used immunohistochemistry and qPCR to confirm the expression of key ARGs with their influences on OS. In conclusion, we identified two ARG-mediated molecular subtypes and a novel seven-ARG risk signature to predict prognoses, immune microenvironments, signal pathways, tumor mutations, and drug sensitivity of PAAD patients.

Distinguishable Prognostic Signatures and Tumor Immunogenicity Between Pancreatic Head Cancer and Pancreatic Body/Tail Cancer.

Background: Pancreatic head cancer and pancreatic body/tail cancer are considered to have different clinical presentations and to have altered outcomes. Methods: Ninety cases of pancreatic adenocarcinoma (PDAC) from our institution were used as a discovery set and 166 cases of PDAC from the TCGA cohort were used as a validation set. According to the anatomical location, the cases of PDAC were divided into the pancreatic head cancer group and the pancreatic body/tail cancer group. Firstly, the different biological functions of the two groups were assessed by ssGSEA. Meanwhile, ESTIMATE and CIBERSORT were conducted to estimate immune infiltration. Then, a novel anatomical site-related risk score (SRS) model was constructed by LASSO and Cox regression. Survival and time-dependent ROC analysis was used to prove the predictive ability of our model in two cohorts. Subsequently, an integrated survival decision tree and a scoring nomogram were constructed to improve prognostic stratification and predictive accuracy for individual patients. In addition, gseaGO and gseaKEGG pathway analyses were performed on genes in the key module by the R package. Results: Overall survival and the objective response rate (ORR) of patients with pancreatic body/tail cancer were markedly superior to those with pancreatic head cancer. In addition, distinct immune characteristics and gene patterns were observed between the two groups. Then, we screened 5 biomarkers related to the prognosis of pancreatic cancer and constructed a more powerful novel SRS model to predict prognosis. Conclusions: Our research shed some light on the revelation of gene patterns, immune and mutational landscape characterizations, and their relationships in different PDAC locations.

Synergistic blocking of RAS downstream signaling and epigenetic pathway in KRAS mutant pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy and lacks effective therapeutic targets. Trametinib is considered to be a promising potential indirectly targeted KRAS inhibitor in PDAC. However, the clinical outcomes were poor. JQ1 displayed a significant synergistic effect when combined with chemotherapy or potential targeted therapy in pancreatic cancer. The impact of Trametinib and JQ1 combination treatment in PDAC remains to be fully elucidated. METHODS: The efficacy of trametinib and JQ1 on cell proliferation and cytotoxicity was assayed in 7 KRAS mutant pancreatic cancer cell lines. The cytotoxic effects of drugs either alone or in combination were evaluated using a luminescent cell viability assay. Immunoblot analysis was carried out to investigate changes in p62 and autophagy. RESULTS: We found that either trametinib or JQ1 alone inhibited the proliferation of some pancreatic cancer cell lines with KRAS alterations, irrespective of the mutational loci of KRAS and the aberrant status of the other driver genes. The synergistic effects of combination treatment of trametinib and JQ1 were observed in both trametinib-resistant and trametinib-sensitive cells. In trametinib-sensitive PDAC cells, the combined treatment definitely inhibited p62 expression compared with trametinib alone, while LC3 expression at high levels changed little. In trametinib-resistant PDAC cells, the combination of MEK/BET inhibitor dramatically decreased p62 expression compared with single agent, while p62 expression increased after anti-autophagic therapy was added. CONCLUSIONS: Blocking RAS downstream signaling and epigenetic pathway synergistically increases the antiproliferative activity in KRAS mutant PDAC cells. Combination therapeutic synergism may induce different cell death modes in different pancreatic cancer subtypes.

Characterization of the genomic landscape in large-scale Chinese patients with pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with an extremely poor prognosis. Effective targets for anticancer therapy confirmed in PDAC are limited. However, the characteristics of genomics have not been fully elucidated in large-scale patients with PDAC from China. METHODS: We collected both blood and tissue samples from 1080 Chinese patients with pancreatic cancer and retrospectively investigated the genomic landscape using next-generation sequencing (NGS). FINDINGS: We found recurrent somatic mutations in KRAS (83.2%), TP53 (70.6%), CDKN2A (28.8%), SMAD4 (23.0%), ARID1A (12.8%) and CDKN2B (8.9%) in Chinese PDAC patients. Compared with primary pancreatic cancers, more genomic alterations accumulated especially cell cycle regulatory gene variants (45.4% vs 31.6%, P < 0.001) were observed in metastatic tumors. The most common mutation site of KRAS is p.G12D (43.6%) in pancreatic cancer. Patients with KRAS mutations were significantly associated with older age and mutations in the other three driver genes, while KRAS wild-type patients contained more fusion mutations and alternative mechanisms of RTK/Ras/MAPK pathway including a number of clinically targetable mutations. KRAS mutations in Chinese cohort were significantly lower than those in Western cohorts (all P < 0.05). A total of 252 (23.3%) patients with the core DNA damage response (DDR) gene mutations were detected. ATM (n =59, 5.5%) was the most frequent mutant DDR gene in patients with pancreatic cancer from China. Patients with germline DDR gene mutations were younger (P = 0.018), while patients with somatic DDR gene mutations were more likely to accumulate in metastatic lesions (P < 0.001) and had higher TMB levels (P < 0.001). In addition, patients with mutant DDR genes and patients carrying TP53 mutation were observed mutually exclusive (P < 0.001). INTERPRETATION: We demonstrated the real-world genomic characteristics of large-scale patients with pancreatic cancer from China which may have promising implications for further clinical significance and drug development. FUNDING: The funders are listed in the Acknowledgement.

EGFR signaling promotes nuclear translocation of plasma membrane protein TSPAN8 to enhance tumor progression via STAT3-mediated transcription.

TSPAN family of proteins are generally considered to assemble as multimeric complexes on the plasma membrane. Our previous work uncovered that TSPAN8 can translocate into the nucleus as a membrane-free form, a process that requires TSPAN8 palmitoylation and association with cholesterol to promote its extraction from the plasma membrane and subsequent binding with 14-3-3θ and importin-ß. However, what upstream signal(s) regulate(s) the nuclear translocation of TSPAN8, the potential function of TSPAN8 in the nucleus, and the underlying molecular mechanisms all remain unclear. Here, we demonstrate that, epidermal growth factor receptor (EGFR) signaling induces TSPAN8 nuclear translocation by activating the kinase AKT, which in turn directly phosphorylates TSPAN8 at Ser129, an event essential for its binding with 14-3-3θ and importin ß1. In the nucleus, phosphorylated TSPAN8 interacts with STAT3 to enhance its chromatin occupancy and therefore regulates transcription of downstream cancer-promoting genes, such as MYC, BCL2, MMP9, etc. The EGFR-AKT-TSPAN8-STAT3 axis was found to be hyperactivated in multiple human cancers, and associated with aggressive phenotype and dismal prognosis. We further developed a humanized monoclonal antibody hT8Ab4 that specifically recognizes the large extracellular loop of TSPAN8 (TSPAN8-LEL), thus being able to block the extraction of TSPAN8 from the plasma membrane and consequently its nuclear localization. Importantly, both in vitro and in vivo studies demonstrated an antitumor effect of hT8Ab4. Collectively, we discovered an unconventional function of TSPAN8 and dissected the underlying molecular mechanisms, which not only showcase a new layer of biological complexity of traditional membrane proteins, but also shed light on TSPAN8 as a novel therapeutic target for refractory cancers.

Prediction of Survival and Tumor Microenvironment Infiltration Based on Pyroptosis-Related lncRNAs in Pancreatic Cancer.

Pancreatic cancer (PC) is a fatal tumor with high mortality. Pyroptosis plays a tumor suppressor role as a novel cell death. However, the influences of the pyroptosis-related lncRNAs (PRlncRNAs) on the prognosis and tumor microenvironment (TME) infiltration have not been fully studied in PC. Using coexpression analysis and univariate Cox regression analysis, we identified seventeen prognostic PRlncRNAs from The Cancer Genome Atlas (TCGA) dataset, which were all expressed differently in normal and tumor samples. A seven-PRlncRNA risk signature was constructed and validated using the least absolute shrinkage and selection operator (LASSO) regression. Furthermore, we verified its independence and created a nomogram to validate the clinical viability of the risk signature. We then identified its relationship with clinical factors and evaluated its values in TME infiltration, functional enrichment, tumor mutation, and therapeutic responses in PC. Lower ImmuneScore, ESTIMATEScore, and advanced tumor stage were connected with high-risk score. The low-risk group was characterized by better OS, elevated immune activation, and higher susceptibility of pazopanib and sunitinib. The high-risk group possessed a worse immune infiltration and poor survival, with higher tumor mutations and lapatinib and paclitaxel that may be better choices in this group. In conclusion, we developed an original seven-PRlncRNA risk signature to predict prognosis, TME infiltration, tumor mutation, and therapeutic options for PC patients.

A Novel Molecular Signature of Cancer-Associated Fibroblasts Predicts Prognosis and Immunotherapy Response in Pancreatic Cancer.

Cancer-associated fibroblasts (CAFs), a prominent population of stromal cells, play a crucial role in tumor progression, prognosis, and treatment response. However, the relationship among CAF-based molecular signatures, clinical outcomes, and tumor microenvironment infiltration remains largely elusive in pancreatic cancer (PC). Here, we collected multicenter PC data and performed integrated analysis to investigate the role of CAF-related genes (CRGs) in PC. Firstly, we demonstrated that α-SMA+ CAFs were the most prominent stromal components and correlated with the poor survival rates of PC patients in our tissue microarrays. Then, we discriminated two diverse molecular subtypes (CAF clusters A and B) and revealed the significant differences in the tumor immune microenvironment (TME), four reported CAF subpopulations, clinical characteristics, and prognosis in PC samples. Furthermore, we analyzed their association with the immunotherapy response of PC patients. Lastly, a CRG score was constructed to predict prognosis, immunotherapy responses, and chemosensitivity in pancreatic cancer patients. In summary, these findings provide insights into further research targeting CAFs and their TME, and they pave a new road for the prognosis evaluation and individualized treatment of PC patients.

SOX9 is a critical regulator of TSPAN8-mediated metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer mainly owing to its proclivity to early metastasis and the lack of effective targeted therapeutic drugs. Hence, understanding the molecular mechanisms underlying early invasion and metastasis by PDAC is imperative for improving patient outcomes. The present study identified that upregulation of TSPAN8 expression in PDAC facilitates metastasis in vivo and in vitro. We found SOX9 as a key transcriptional regulator of TSPAN8 expression in response to EGF stimulation. SOX9 modulation was sufficient to positively regulate endogenous expression of TSPAN8, with concomitant in vitro phenotypic changes such as loss of cell-matrix adherence and increased invasion. Moreover, increased SOX9 and TSPAN8 levels were shown to correlate in human pancreatic cancer specimens and downregulated in vitro by EGFR tyrosine kinase inhibitors. High expression of SOX9 and TSPAN8 has been associated with tumor stage, poor prognosis and poor patient survival in PDAC. In conclusion, this study highlights the importance of the EGF-SOX9-TSPAN8 signaling cascade in the control of PDAC invasion and implies that TSPAN8 may be a promising novel therapeutic target for the treatment of PDAC.

ZGRF1 Is Associated with Poor Prognosis in Triple-Negative Breast Cancer and Promotes Cancer Stemness Based on Bioinformatics.

BACKGROUND: Recently, long non-coding RNAs (lncRNAs) are important populations of non-coding RNAs with defined key roles in normal breast development as well as breast tumorigenesis. Triple-negative breast cancer (TNBC) is a particular breast cancer subtype with poor prognosis because of highly invasive and no specific drug treatment yet. Breast cancer stems cells (BCSCs) cause a high risk of invasion, metastasis and drug resistance for breast cancer patients. METHODS: Two microarrays of BCSCs and no-BCSCs were isolated from mammosphere-3D-cultured MCF-7 cells, differentially expressed lncRNAs (DELs) were screened out by Gene Expression Omnibus (GEO). Gene ontology enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also performed to analyze DELs features. Using the STRING database to analyze DELs interaction network module to further screen the hub lncRNAs related to tumor stemness and make functional annotations. The expressions of hub DELs were validated using data from The Cancer Genome Atlas database. In addition, the expression analysis and survival analysis were conducted using GEO was utilized to analyze DELs in TNBC using GEPIA database. RESULTS: A total of 143 aberrantly expressed lncRNAs in BCSCs were identified, and 25 lncRNAs were downregulated and 118 lncRNAs were upregulated compared to non-BCSCs. Up- and downregulated top 3 lncRNAs were selected and verified by RT-PCR. Notably, GO enrichment analysis and KEGG pathway analysis indicated that RNA transport, spliceosome, oxidative phosphorylation, NOD-like receptor signaling pathway, PI3K-Akt signaling pathway, and metabolic pathways may serve important roles in BCSCs. Additionally, the function loss assay indicated that ZGRF1 positively upregulated phenotype and biological functions of BCSCs in vitro. Collectively, our work establishes the lncRNAs signature in BCSCs and these findings assess us with evidence to explore further functionalities of lncRNAs in BCSCs and provide a novel therapeutic strategy for breast cancer. CONCLUSION: Our work establishes the lncRNAs signature in BCSCs and these findings assess us with evidence to explore further functionalities of lncRNAs in BCSCs and provide a novel therapeutic strategy for breast cancer. ZGRF1 expression is upregulated in TNBC patients and has a poor prognosis, which can be promising biomarkers.

TSPAN8 promotes cancer cell stemness via activation of sonic Hedgehog signaling.

Cancer stem cells (CSCs) represent a major source of treatment resistance and tumor progression. However, regulation of CSCs stemness is not entirely understood. Here, we report that TSPAN8 expression is upregulated in breast CSCs, promotes the expression of the stemness gene NANOG, OCT4, and ALDHA1, and correlates with therapeutic resistance. Mechanistically, TSPAN8 interacts with PTCH1 and inhibits the degradation of the SHH/PTCH1 complex through recruitment of deubiquitinating enzyme ATXN3. This results in the translocation of SMO to cilia, downstream gene expression, resistance of CSCs to chemotherapeutic agents, and enhances tumor formation in mice. Accordingly, expression levels of TSPAN8, PTCH1, SHH, and ATXN3 are positively correlated in human breast cancer specimens, and high TSPAN8 and ATXN3 expression levels correlate with poor prognosis. These findings reveal a molecular basis of TSPAN8-enhanced Sonic Hedgehog signaling and highlight a role for TSPAN8 in promoting cancer stemness.