Overexpression Of Twist Research Articles

Abstract B008: Twist1 drives pancreatic ductal adenocarcinoma-mediated muscle cachexia

Abstract Cancer cachexia is a debilitating syndrome that affects the survival and quality of life of cancer patients. Up to 95% of pancreatic ductal adenocarcinoma (PDAC) patients experience muscle cachexia, yet the underlying mechanisms remain poorly understood. The transcription factor Twist1 plays essential roles in mesenchymal stem cell fate determination, and its deregulation contributes to cancer progression and metastasis. In this study, we report on the serendipitous finding that Twist1 functions in PDAC to drive muscle cachexia. We found that conditional overexpression of Twist1 in the pancreatic epithelium (pTwist1) during early development resulted in severe growth retardation, which was mainly characterized by muscle hypotrophy. This intriguing observation prompted us to inquire whether pTwist1 could function in the pancreatic epithelium to regulate muscle mass, and if so, whether this has any implication as it relates to PDAC-mediated muscle cachexia. We found that enforced expression of pTwist1 in healthy adult animals was sufficient to elicit the cardinal features of muscle cachexia, including progressive decline in body weight, physical activity, muscle force, and muscle mass. In the context of PDAC, enforced expression of pTwist1 in a KrasG12D background led to a dramatic acceleration of tumor progression, and this was associated with severe muscle cachexia. To the best of our knowledge, these findings provide the first evidence that the pro-malignant transcription factor Twist1 functions in PDAC to drive muscle cachexia, thus revealing new insights into the underlying mechanisms of muscle that could be harnessed to curb this debilitating syndrome. Citation Format: Parash Parajuli, Azeddine Atfi. Twist1 drives pancreatic ductal adenocarcinoma-mediated muscle cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B008.

Immunohistochemical overexpression of TWIST1 in metastatic melanomas compared with primary melanomas and nevi

Melanoma is an aggressive neoplasia with poor prognosis when not identified and treated early. Phenotypic changes related to melanocytic cell invasion and metastasis have been associated with overexpression of the transcription factor TWIST1, which contributes to upregulation of epithelial-mesenchymal transition in melanoma. We aimed to evaluate the immunohistochemical expression of TWIST1 in nevi, primary melanomas and metastatic melanomas. This is a retrospective cross-sectional study of 71 histological samples with diagnosis of nevus, primary melanoma and metastatic melanoma. The immunohistochemical analysis was performed applying Twist1 ‘clone 2C1a’ monoclonal antibody and the results were interpreted according to scores of intensities and percentage of positive cells. There was TWIST1 nuclear overexpression in metastatic melanomas when compared to the primary melanomas and melanocytic nevi groups. There was minor significant difference regarding nuclear expression between primary melanomas and nevi. In conclusion, the results of our evaluation of immunohistochemical expression can be consistent with the role of the TWIST1 in metastasis of melanomas.

Restraint of melanoma progression by cells in the local skin environment.

Keratinocytes, the dominant cell type in the melanoma microenvironment during tumor initiation, exhibit diverse effects on melanoma progression. Using a zebrafish model of melanoma and human cell co-cultures, we observed that keratinocytes undergo an Epithelial-Mesenchymal Transition (EMT)-like transformation in the presence of melanoma, reminiscent of their behavior during wound healing. Surprisingly, overexpression of the EMT transcription factor Twist in keratinocytes led to improved overall survival in zebrafish melanoma models, despite no change in tumor initiation rates. This survival benefit was attributed to reduced melanoma invasion, as confirmed by human cell co-culture assays. Single-cell RNA-sequencing revealed a unique melanoma cell cluster in the Twist-overexpressing condition, exhibiting a more differentiated, less invasive phenotype. Further analysis nominated homotypic jam3b-jam3b and pgrn-sort1a interactions between Twist-overexpressing keratinocytes and melanoma cells as potential mediators of the invasive restraint. Our findings suggest that EMT in the tumor microenvironment (TME) may limit melanoma invasion through altered cell-cell interactions.

The oncogenic roles of GPR176 in ovarian cancer: a molecular target for aggressiveness and gene therapy

Background At present, the discovery of new biomarkers is of great significance for the early diagnosis, treatment and prognosis assessment of ovarian cancer. Previous findings indicated that aberrant G-protein-coupled receptor 176 (GPR176) expression might contribute to tumorigenesis and subsequent progression. However, the expression of GPR176 and the molecular mechanisms in ovarian cancer had not been investigated. Methods GPR176 expression was compared with clinicopathological features of ovarian cancer using immunohistochemical and bioinformatics analyses. GPR176-related genes and pathways were analysed using bioinformatics analysis. Additionally, the effects of GPR176 on ovarian cancer cell phenotypes were investigated. Results GPR176 expression positively correlated with elder age, clinicopathological staging, tumour residual status, and unfavourable survival of ovarian cancer, but negatively with purity loss, infiltration of B cells, and CD8+ T cells. Gene Set Enrichment Analysis showed that differential expression of GPR176 was involved in focal adhesion, ECM-receptor interaction, cell adhesion molecules and so on. STRING and Cytoscape were used to determine the top 10 nodes. Kyoto Encyclopaedia of Genes and Genomes analysis indicated that GPR176-related genes were involved in the ECM structural constituent and organisation and so on. GPR176 overexpression promoted the proliferation, anti-apoptosis, anti-pyroptosis, migration and invasion of ovarian cancer cells with overexpression of N-cadherin, Zeb1, Snail, Twist1, and under-expression of gasdermin D, caspase 1, and E-cadherin. Conclusion GPR176 might be involved in the progression of ovarian cancer. It might be used as a biomarker to indicate the aggressive behaviour and poor prognosis of ovarian cancer and a target of genetic therapy.

TWIST1 Drives Cytotoxic CD8+ T-Cell Exhaustion through Transcriptional Activation of CD274 (PD-L1) Expression in Breast Cancer Cells.

In breast cancer, epithelial-mesenchymal transition (EMT) is positively associated with programmed death ligand 1 (PD-L1) expression and immune escape, and TWIST1 silences ERα expression and induces EMT and cancer metastasis. However, how TWIST1 regulates PD-L1 and immune evasion is unknown. This study analyzed TWIST1 and PD-L1 expression in breast cancers, investigated the mechanism for TWIST1 to regulate PD-L1 transcription, and assessed the effects of TWIST1 and PD-L1 in cancer cells on cytotoxic CD8+ T cells. Interestingly, TWIST1 expression is correlated with high-level PD-L1 expression in ERα-negative breast cancer cells. The overexpression and knockdown of TWIST1 robustly upregulate and downregulate PD-L1 expression, respectively. TWIST1 binds to the PD-L1 promoter and recruits the TIP60 acetyltransferase complex in a BRD8-dependent manner to transcriptionally activate PD-L1 expression, which significantly accelerates the exhaustion and death of the cytotoxic CD8+ T cells. Accordingly, knockdown of TWIST1 or BRD8 or inhibition of PD-L1 significantly enhances the tumor antigen-specific CD8+ T cells to suppress the growth of breast cancer cells. These results demonstrate that TWIST1 directly induces PD-L1 expression in ERα-negative breast cancer cells to promote immune evasion. Targeting TWIST1, BRD8, and/or PD-L1 in ERα-negative breast cancer cells with TWIST1 expression may sensitize CD8+ T-cell-mediated immunotherapy.

Polyphyllin I Mitigated IL-1β-Induced Chondrocytes Damage through Downregulating TWIST1 Expression.

Osteoarthritis (OA) is a chronic joint disease characterized by the degradation of articular cartilage. Polyphyllin I (PPI) has anti-inflammatory effects in many diseases. However, the mechanism of PPI in OA remains unclear. <P> </P> Methods: HC-a cells treated with IL-1β were identified by immunofluorescence staining and microscopic observation. The expression of collagen II and DAPI in HC-a cells was detected by immunofluorescence. The effects of gradient concentration of PPI on IL-1β-induced cell viability, apoptosis, senescence, and inflammatory factor release were detected by MTT, flow cytometry, SA-β-Gal assay and ELISA, respectively. Expressions of apoptosis-related genes, extracellular matrix (ECM)- related genes, and TWIST1 were determined by qRT-PCR and western blot as needed. The above-mentioned experiments were conducted again after TWIST1 overexpression in IL-1β-induced chondrocytes. <P> </P> Results: IL-1β reduced the number of chondrocytes and the density of collagen II. PPI (0.25, 0.5, 1 µmol/L) had no effect on cell viability, but it dose-dependently elevated the inhibition of cell viability regulated by IL-1β. The elevation of cell apoptosis, senescence and expression of IL-6 and TNF-α were suppressed by PPI in a dosedependent manner. Additionally, PPI reduced the expression of cleaved caspase-3, bax, MMP-3, and MMP-13 and promoted the expression of collagen II. TWIST1 expression was diminished by PPI. TWIST1 overexpression reversed the abovementioned effects of PPI on chondrocytes. <P> </P> Conclusion: PPI suppressed apoptosis, senescence, inflammation, and ECM degradation of OA chondrocytes by downregulating the expression of TWIST1.

Epithelial-mesenchymal transition dysregulation in prostate cancer: Insights from molecular unraveling and epidemiological analyses in Tunisia, North Africa.

The progression of prostate cancer (PCa) has been linked worldwide, including in African populations, to the dysregulation of the epithelial-mesenchymal transition (EMT). To clarify the connection among EMT markers, clinicopathological parameters, and epidemiological factors, we analyzed 35 PCa specimens from patients in Tunisia, a country in North Africa, arranged by stages. We also carried out extensive molecular and epidemiological analyses. Significant dysregulation of EMT genes was found, with an overexpression of ZEB-1, Twist, Snail-1, and Vimentin (p<0.05) and underexpression of E-cadherin and β-catenin (p<0.05). Positive correlations were observed between transcription factors and the mesenchymal marker Vimentin (p<0.001, r=0.574; p=0.029, r=0.411; and p<0.001; r=0.506) according to Spearman correlation analyses, whereas negative correlations were found between epithelial markers (E-cadherin, β-catenin) and Vimentin (p<0.05; r<0). Higher PSA, Gleason scores, and metastasis were all correlated with the dysregulation of EMT (p<0.05). Notably, there was a positive correlation between higher consumption of tobacco (≥20 Packets per year) and Vimentin expression (p<0.001, r=0.854), suggesting a relationship between smoking and EMT activation in the Tunisian population. Moreover, Twist showed a positive correlation with diabetes (p<0.001, r=0.385), whereas no significant correlations were found between EMT markers and comorbidities such as hypertension and coronary insufficiency. These results demonstrate the intricate connection between molecular changes, epidemiological factors, and disease progression, and they emphasize the crucial role that EMT plays in promoting PCa aggressiveness in African populations, particularly in Tunisia. In summary, understanding these correlations could help develop focused treatment plans and enhance patient outcomes for PCa management in African settings.

Abstract 1438: Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response

Abstract In this study, we investigated the contributions of the epithelial-mesenchymal transition (EMT) program to small cell lung cancer (SCLC) tumorigenesis and chemoradiation (CRT) response. SCLC is a highly aggressive and deadly neuroendocrine malignancy. Two major factors that contribute to the high mortality of SCLC are (1) early metastasis and (2) rapid development of therapy resistance. Recent research suggests upregulation of EMT and the EMT transcription factor Twist1 correlated with accelerated tumor progression and CRT resistance in SCLC. Due to the lack of suitable animal models, the causal relationship between the EMT program and SCLC tumorigenesis and therapy response has not been rigorously investigated. To this end, we have generated a novel genetically engineered mouse model (GEMM) termed RPGT (Rb1Flox; Trp53Flox; ROSA26LSL-rtTA-IRES-EGFP; Twist1-TetO7-Luc). The RPGT GEMM enables the generation of autochthonous SCLC tumors after induction with Cre recombinase adenovirus. By withdrawing or providing doxycycline to mice, we can control Twist1 expression to activate or inactivate EMT in tumors. Upon characterizing mouse tumor samples with histological and molecular analyses, we observed that both control (no Twist1 overexpression) and Twist1-overexpressing RPGT mice developed neuroendocrine SCLC tumors. While SCLC-A was the predominant subtype in both cohorts, RPGT tumors exhibited more heterogeneity. Importantly, although no difference in tumorigenesis was observed, Twist1 overexpression dramatically increased the metastatic incidence in RPGT (87-100%) compared to control (25-50%) animals, indicating an important role of EMT in SCLC dissemination. Furthermore, transcriptomic profiling of primary tumors and matching metastases in RPGT mice revealed downregulation of Twist1 and EMT in metastases, suggesting that EMT suppression was necessary for metastatic outgrowth. To evaluate the impact of EMT activation on SCLC sensitivity to CRT, we performed in vitro viability assays on primary tumor cell lines established from RPGT tumors. We found that repressing Twist1 expression enhanced SCLC susceptibility to CRT. To validate these results in vivo, we treated RPGT mice with vs. without Twist1 overexpression with CRT. Consistent with our in vitro results, our in vivo data indicated that Twist1 overexpression promoted CRT resistance in SCLC tumors. Overall, our data suggest that the EMT program plays an important role in SCLC differentiation, metastasis, and resistance against chemo-radiotherapy. Citation Format: Triet Nguyen, Jinhee Chang, Kathleen Gabrielson, Amol Shetty, Yang Song, Audrey Lafargue, Shreya Jagtap, Danielle Council, Aaron Chan, Dipanwita Dutta Chowdhury, Muhammad Ajmal Khan, Nick Connis, Francesca Carrieri, Eddie Imada, Daniel Sforza, Eric Gardner, Christopher McFarland, Luigi Marchionni, Mohammad Rezaee, Christine Hann, Phuoc Tran. Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1438.

Abstract 1444: Modeling Twist1 overexpression in a pancreatic ductal adenocarcinoma mouse model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer characterized by very poor survival outcomes. Genomic sequencing of human PDAC tissues and cell lines have identified four molecular subtypes: quasimesenchymal (QM), pancreatic progenitor, immunogenic, and aberrantly differentiated endocrine exocrine (ADEX). QM-PDAC has the worse prognosis, but no in vivo models exist for this molecular subtype. Our preliminary analysis using publicly available data demonstrated that the epithelial-to-mesenchymal transition (EMT) transcription factor TWIST1 is upregulated in QM-PDAC. We hypothesized that the TWIST1-dependent plasticity program was a key regulator of QM-PDAC subtype development. We created a tetracycline-inducible tissue specific Twist1 PDAC genetically engineered mouse model (GEMM). Tumor development in the mouse pancreatic ductal epithelium was directed using the Pdx1 promoter-Cre (P) allele leading to stable native expression of the LSL-KrasG12D oncogene (R) and LSL-rtTA-IRES-GFP (G) alleles. The non-Twist1 GEMM (PGR) was crossed with the Twist1-tetO-Luc (T) mouse to create the PGRT model which allows inducible expression of Twist1 and luciferase in pancreatic epithelium under doxycycline treatment (Tet-ON system). Tumor progression, lineage tracing and metastatic invasion are followed by fluorescence (GFP) and bioluminescence (BLI) imaging. The characterization of this novel PGRT mouse model showed decreased overall survival compared to control non-Twist1 containing mice (~35 weeks PGRT (n=17) vs ~60 weeks PGR (n=10)). Histological and immunohistochemistry analyses showed the PGRT pancreatic tumors resemble the QM-PDAC subtype and vimentin overexpression correlates with Twist1-dependent induction of EMT. We attempted to confirm the QM-PDAC profile via cellular markers: GATA6-, HNF1A-, KRT81+, KRT17+, KRT20+, P63+ and ∆P63+. The PGR and standard KPC (Kras+/LSL-G12D; Trp53+LSL-R172H; Pdx1-Cre) PDAC model were also profiled. We observed that 75% of the PGRT tumors displayed a QM-PDAC profile, contrasting with the absence of such profile in both PGR and KPC tumors. Examining the tumors from PGR and KPC, 100% of the PGR and 72.27% KPC tumors exhibited a non-squamous PDAC profile while an intermediate squamous profile was observed in 25% PGRT tumors and 27.27% KPC tumors. Furthermore, Twist1 induction resulted in intra-abdominal and lung metastases. We observed that 87.5% of the PGRT mice were positive for metastases while we observed no positivity for PGR mice and 63.64% positivity in KPC mice. In conclusion, these data suggest that Twist1 overexpression cooperates with KrasG12D for PDAC tumor development mimicking the human QM-PDAC subtype to promote progression towards metastasis and early lethality. Potential uses of this novel model include testing treatment strategies to ultimately improve patient outcomes with QM-PDAC. Citation Format: Muhammad Ajmal Khan, Jinhee Chang, Triet Nguyen, Danielle N. Council, Dipanwita Dutta Chowdhury, Amol Shetty, Yang Song, Aaron Chan, Christine Lam, Priyanshi Patel, Lucas Tran, Jiayu Chen, Katriana Nugent, Matthew Ballew, Ghali Lemtiri-Chlieh, Francesca A. Carrieri, Reem Malek, Hailun Wang, Noelle R. Thielman, Keyu Li, Lei Zheng, Kathleen Gabrielson, Phuoc T. Tran, Audrey Lafargue. Modeling Twist1 overexpression in a pancreatic ductal adenocarcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1444.

Abstract 4743: Targeting a novel TWIST1-Hexokinase II pathway to overcome MET TKI resistance in NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the most common primary tumor to metastasize to the brain, with up to 40% of NSCLC patients developing brain metastases (BM). These BM portend a poor prognosis, even when extracranial disease is well-controlled. While targeted therapy and immunotherapy have improved lung cancer survival, there are no therapies specifically targeting NSCLC BM. Our lab identified a significant enrichment of MET amplification in lung adenocarcinoma (LUAD) BM (16%) compared to primary NSCLC (3%) and liver metastases (5%). MET, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), promote proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and metastasis. Although MET tyrosine kinase inhibitors (TKIs) are approved for use in METaltered NSCLC, half of patients with MET alterations fail to respond or inevitably acquire TKI resistance. We have found that MET pathway activation by MET alterations leads to increased expression of TWIST1, an EMT transcription factor required for tumorigenesis in MET-dependent NSCLC. Together, the HGF/MET/TWIST1 axis promotes cancer survival and metastasis. We found that MET amplified BM had distinct transcriptional signatures reflecting increased glycolysis. A similar shift toward glycolysis was observed in the MET amplified metastatic LUAD cell line (H1993) compared to the MET wild type primary LUAD line (H2073) derived from the same patient. Furthermore, LUAD cell lines with high MET expression demonstrated increased expression and activity of glycolytic enzymes, as well as increased susceptibility to glucose deprivation and glycolytic inhibitors (2DG, PFK158, AT-101, V-9302). Conversely, treatment of MET amplified cells with MET TKI, capmatinib, reduced glycolysis and oxidation phosphorylation to the levels observed in MET wild type LUAD lines. Activation of the MET pathway with HGF or TWIST1 overexpression specifically increased the mRNA and protein expression of the key glycolytic enzyme, Hexokinase II (HK2), compared to other glycolytic pathway proteins. Conversely, inhibition of the MET pathway with capmatinib or the TWIST1 inhibitor, harmine, decreased HK2 protein expression through a MYC-independent pathway. However, TWIST1 was required for MET-dependent transcriptional upregulation of HK2. Remarkably, in two novel MET altered models (cell line and patient derived xenograft) of acquired MET TKI resistance, TWIST1 and HK2 expression were increased at the time of resistance. Both models remained sensitive to pharmacologic inhibition of either TWIST1 or glycolysis at the time of acquired resistance to MET TKIs. These findings suggest that continued upregulation of the TWIST1-HK2 axis is required for acquired MET TKI resistance. In summary, these studies suggest a targetable, metabolic reprogramming in MET altered LUAD BM mediated through a novel TWIST1-HK2 pathway. Citation Format: Purva H. Rumde, Kasey R. Cargill, Vinod Kumar, Princey Devadassan, Jason Chen, Leviticus McGraw-Sapp, Xiaxuan Wu, Sanja Dacic, Riyue Bao, Bharathri Sivakama, Eric S. Goetzman, Sameer Agnihotri, Steven J. Mullett, Stacey G. Wendell, Laura P. Stabile, Timothy F. Burns. Targeting a novel TWIST1-Hexokinase II pathway to overcome MET TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4743.

Interaction between Wnt/β-catenin signaling pathway and EMT pathway mediates the mechanism of sunitinib resistance in renal cell carcinoma

BackgroundTargeted drugs are the main methods of RCC treatment. However, drug resistance is common in RCC patients, in-depth study of the drug-resistant mechanism is essential.MethodsWe constructed sunitinib resistant and Twist overexpressed A498 cells, and studied its mechanisms in vitro and in vivo.ResultsIn cell research, we found that either sunitinib resistance or Twist overexpression can activate Wnt/β-catenin and EMT signaling pathway, and the sunitinib resistance may work through β-catenin/TWIST/TCF4 trimer. In zebrafish research, we confirmed the similarity of Twist overexpression and sunitinib resistance, and the promoting effect of Twist overexpression on drug resistance.ConclusionsSunitinib resistance and Twist overexpression can activate Wnt/β-catenin signaling pathway and EMT to promote the growth and metastasis of RCC cells.

Deciphering the interaction between Twist1 and PPARγ during adipocyte differentiation

Obesity, a worldwide epidemic in recent years, is mainly due to the uncontrolled development of adipose tissues, which includes adipocyte hypertrophy and hyperplasia. Adipocyte differentiation is a process involving multiple transcription factor cascades, and the exact mechanism has not yet been defined. As a bHLH transcription factor, Twist1 exerts its activity by forming homo- or heterodimers with other factors. In this study, we showed Twist1 restricts adipogenesis through PPARγ. Expression of various differentiation markers (including PPARγ and adiponectin) and triglyceride-containing lipid droplets were decreased with overexpression of Twist1. Pathway enrichment analysis of RNA-seq data showed that differentially expressed genes (DEGs) caused by Twist1 overexpression were significantly related to lipolysis and PPARγ signaling. This implicates that Twist1 plays important regulatory roles in these processes. ChIP and dual luciferase assays showed that Twist1 could bind either PPARγ or adiponectin promoter to repress their respective transcription or directly to PPARγ protein to regulate its transcriptional activity. Furthermore, Twist1 directly interacted RXRα, which usually forms heterodimer with PPARγ to regulate adipogenesis. Taken together, our results suggest that Twist1 is an inhibitory modulator of adipogenesis and its function is likely through direct interaction with PPARγ protein or its gene promoter.

Expression profiling of extramedullary acute myeloid leukemia suggests involvement of epithelial-mesenchymal transition pathways.

Extramedullary (EM) colonization is a rare complication of acute myeloid leukemia (AML), occurring in about 10% of patients, but the processes underlying tissue invasion are not entirely characterized. Through the application of RNAseq technology, we examined the transcriptome profile of 13 AMLs, 9 of whom presented an EM localization. Our analysis revealed significant deregulation within the extracellular matrix (ECM)-receptor interaction and focal-adhesion pathways, specifically in the EM sites. The transcription factor TWIST1, which is known to impact on cancer invasion by dysregulating epithelial-mesenchymal-transition (EMT) processes, was significantly upregulated in EM-AML. To test the functional impact of TWIST1 overexpression, we treated OCI-AML3s with TWIST1-siRNA or metformin, a drug known to inhibit tumor progression in cancer models. After 48 h, we showed downregulation of TWIST1, and of the EMT-related genes FN1 and SNAI2. This was associated with significant impairment of migration and invasion processes by Boyden chamber assays. Our study shed light on the molecular mechanisms associated with EM tissue invasion in AML, and on the ability of metformin to interfere with key players of this process. TWIST1 may configure as candidate marker of EM-AML progression, and inhibition of EMT-pathways may represent an innovative therapeutic intervention to prevent or treat this complication.

LncRNA LZTS1-AS1 induces proliferation, metastasis and inhibits autophagy of pancreatic cancer cells through the miR-532 /TWIST1 signaling pathway

The 5 year survival rate after diagnosis of pancreatic cancer (PANC) is less than 5%, and it is one of the malignant tumors with the worst prognosis. Identification of novel oncogenes involved in the occurrence of pancreatic cancer is of great significance to improve the overall survival of PANC patients. Our previous study found that miR-532 is a key factor in PANC occurrence and development, and this study further explored its mechanism. We found that the expression of lncRNA LZTS1-AS1 was elevated in PANC tumor tissues and cells, and correlated with poor prognosis. In vitro experiments confirmed that LZTS1-AS1 could promote proliferation, oncogenicity, migration, and invasion of PANC cells, and inhibit apoptosis and autophagy. However, miR-532 had the completely opposite effect, and inhibition of miR-532 counteracted the effect of LZTS1-AS1 on PANC cells. Dual luciferase gene reporter assay and RNA immunoprecipitation assay confirmed the targeting relationship between LZTS1-AS1 and miR-532, and their expression levels were negatively correlated in PANC tissues. Overexpression of TWIST1 could counteract the effect of miR-532 in PANC cells, and the expression levels of both were negatively changed in PANC tissues and cells. Our results suggest that lncRNA LZTS1-AS1 acts as an oncogene to promote the metastasis of PANC and inhibit autophagy, and its mechanism may be to regulate TWIST1 through sponge miR-532. This study provides novel biomarkers and therapeutic targets for PANC.

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity.

In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

LncRNA FOXD2-AS1 facilitates the progression of hepatocellular carcinoma by regulating TWIST1.

The study aimed at elucidating the role of FOXD2-AS1 in facilitating the malignant progression of hepatocellular carcinoma (HCC) by regulating TWIST1. Relative levels of FOXD2-AS1 and TWIST1 in HCC tissues classified by tumor size and tumor node metastasis (TNM) staging were detected by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The Kaplan-Meier method was applied to assess the prognostic potential of FOXD2-AS1 in HCC patients, followed by survival rate comparison using a log-rank test. After the knockdown of FOXD2-AS1 in HepG2 cells, the viability and migratory abilities were examined by Cell Counting Kit-8 (CCK-8) and transwell assay, respectively. The subcellular distribution of FOXD2-AS1 was detected. Finally, the involvement of TWIST1 in the regulation of HCC cell functions influenced by FOXD2-AS1 was explored. FOXD2-AS1 was upregulated in HCC tissues, especially large tumor size or stage III-IV cases. High levels of FOXD2-AS1 predicted poor prognosis in HCC patients. FOXD2-AS1 was mainly distributed in the nucleus, and knockdown of FOXD2-AS1 weakened proliferative and migratory abilities in HepG2 cells. TWIST1 was upregulated in HCC tissues, which was positively correlated to FOXD2-AS1 level. The overexpression of TWIST1 could reverse the inhibited proliferative and migratory abilities in HepG2 cells with FOXD2-AS1 knockdown. FOXD2-AS1 facilitates the progression of HCC by upregulating TWIST1.

Abstract 3853: The TWIST1-p27 signaling pathway is a critical determinant of MET TKI cell cycle arrest and resistance in MET altered non-small cell lung cancer

Abstract Advancement in classifying NSCLC into molecularly defined subgroup that respond to specific therapies have shifted the treatment paradigm towards personalized targeted therapy approach. A major obstacle for the treatment of oncogene driven non-small cell lung cancer (NSCLC) patients is both de novo and acquired resistance to targeted therapies. Approximately 4-6% of NSCLC patients harbor MET amplifications/mutations which are important targetable oncogenic drivers in the clinic. In addition, MET amplification has also emerged as a common mechanism of acquired resistance to EGFR TKIs as well as other targeted therapies. Crizotinib, tepotinib and capmatinib are FDA approved MET TKIs for MET exon 14 skipping mutant NSCLC and have shown activity against MET amplified NSCLC as well. Despite high response rates with these newer TKIs, the majority of patients with targetable MET alterations fail to respond and acquired resistance is inevitable. Multiple molecular mechanisms of MET TKIs resistance encompassing both on-target (HGF overexpression) and bypass have been identified. Furthermore, at the time of resistance, an epithelial-to-mesenchymal (EMT) phenotype is often observed. We have found that HGF/MET pathway is a major regulator of the TWIST1, TWIST1 is required for MET altered NSCLC and cooperatives with HGF in vivo during tumorigenesis. Here, we reported that EMT transcription factor TWIST1 mediates a bypass of a MET TKI-mediated p27-dependent growth arrest. To study the mechanism of TKI resistance in MET altered NSCLC, we used multiple MET dysregulated human cell lines (H1437, H596, H1993, and H1648), a novel acquired resistance model of MET TKI resistance (H1993-CR cells), two syngeneic tobacco carcinogen induced mouse lung cell lines (FVBW-17 and FVBCH-17), and three different animal models (mouse xenografts, PDXs, transgenic animals). We found that TWIST1 overexpression led to MET TKI resistance in vitro and in vivo in MET altered cell lines by overcoming cell cycle arrest. This was accompanied by a failure to induce p27 in the presence of TWIST1 overexpression. Similarly, loss of p27 induction and G1 arrest was also observed in our model of acquired MET TKI resistance. Conversely, TWIST1 inhibition restore sensitivity to TKIs in vitro and in vivo and lead to induction of p27. p27 appears to be require for MET TKI induced cell cycle arrest and sensitivity as silencing p27 lead to MET TKI resistance. Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in MET-driven NSCLC. Citation Format: Vinod Kumar, Zachary A. Yochum, Princey Devadassan, Eric Huang, Ethan Miller, Vasavi Ayyala, Purva H. Rumde, Roja Baruwal, James O’Brien, Laura P. Stabile, Timothy F. Burns. The TWIST1-p27 signaling pathway is a critical determinant of MET TKI cell cycle arrest and resistance in MET altered non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3853.

In vitro and in vivo anti-tumor activity of Coenzyme Q0 against TWIST1-overexpressing HNSCC cells: ROS-mediated inhibition of EMT/metastasis and autophagy/apoptosis induction

HNSCC (Head and Heck Squamous Cell Carcinoma) is a reasonably prevalent cancer with a high mortality rate. In this study, we tried to examine the anti-metastasis and apoptosis/autophagy actions of Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone), a derivative of Antrodia camphorata in HNCC TWIST1 overexpressing (FaDu-TWIST1) cells as well as in vivo tumor xenograft mice model. Using fluorescence based cellular assays, western blot and nude mice tumor xenografts, we determined that CoQ0 effectively reduced cell viability and displayed rapid morphological changes in FaDu-TWIST1 cells compared to FaDu cells. Non/sub-cytotoxic concentrations of CoQ0 treatment reduces the cell migration by downregulating TWIST1 and upregulating E-cadherin. Apoptosis produced by CoQ0 was mostly related with caspase-3 activation, PARP cleavage, and VDAC-1 expression. The FaDu-TWIST1 cells treated with CoQ0 exhibits autophagy-mediated LC3-II accumulation and acidic vesicular organelles (AVOs) formation. Pre-treatment with 3-MA and CoQ effectively prevented CoQ0-induced cell death and CoQ0-triggered autophagy in FaDu-TWIST cells as a death mechanism. CoQ0 induces ROS production in FaDu-TWIST1 cells and NAC pre-treatment significantly reduces anti-metastasis, apoptosis, and autophagy. Likewise, ROS-mediated AKT inhibition regulates CoQ0-induced apoptosis/autophagy in FaDu-TWIST1 cells. In vivo studies exhibit, CoQ0 effectively delays and reduces the tumor incidence and burden in FaDu-TWIST1-xenografted nude mice. Current findings display, CoQ0 exhibits a novel anti-cancer mechanism hence, it might be appropriate for anticancer therapy, and a new potent drug for HNSCC.

TGFβ instructs mTORC2 to activate PKCβII for increased TWIST1 expression in proximal tubular epithelial cell injury.

The plasticity of proximal tubular epithelial cells in response to TGFβ contributes to the expression of TWIST1 to drive renal fibrosis. The mechanism of TWIST1 expression is not known. We show that both PI3 kinase and its target mTORC2 increase TGFβ-induced TWIST1 expression. TGFβ enhances phosphorylation on Ser-660 in the protein kinase C βII (PKCβII) hydrophobic motif site. Remarkably, phosphorylation-deficient PKCβIIS660A, kinase-dead PKCβII, and PKCβII knockdown blocked TWIST1 expression by TGFβ. Inhibition of TWIST1 arrested TGFβ-induced tubular cell hypertrophy and the expression of fibronectin, collagen I (α2), and α-smooth muscle actin. By contrast, TWIST1 overexpression induced these pathologies. Interestingly, the inhibition of PKCβII reduced these phenomena, which were countered by the expression of TWIST1. These results provide the first evidence for the involvement of the mTORC2-PKCβII axis in TWIST1 expression to promote tubular cell pathology.

TWIST1 Plays Role in Expression of Stemness State Markers in ESCC

Stemness markers play critical roles in the maintenance of key properties of embryonic stem cells (ESCs), including the pluripotency, stemness state, and self-renewal capacities, as well as cell fate decision. Some of these features are present in cancer stem cells (CSCs). TWIST1, as a bHLH transcription factor oncogene, is involved in the epithelial-mesenchymal transition (EMT) process in both embryonic and cancer development. Our aim in this study was to investigate the functional correlation between TWIST1 and the involved genes in the process of CSCs self-renewal in human esophageal squamous cell carcinoma (ESCC) line KYSE-30. TWIST1 overexpression was enforced in the ESCC KYSE-30 cells using retroviral vector containing the specific pruf-IRES-GFP-hTWIST1 sequence. Following RNA extraction and cDNA synthesis, the mRNA expression profile of TWIST1 and the stem cell markers, including BMI1, CRIPTO1, DPPA2, KLF4, SOX2, NANOG, and MSI1, were assessed using relative comparative real-time PCR. Ectopic expression of TWIST1 in KYSE-30 cells resulted in an increased expression of TWIST1 compared to control GFP cells by nearly 9-fold. Transduction of TWIST1-retroviral particles caused a significant enhancement in BMI1, CRIPTO1, DPPA2, KLF4, and SOX2 mRNA expression, approximately 4.5-, 3.2-, 5.5-, 3.5-, and 3.7-folds, respectively, whereas this increased TWIST1 expression caused no change in the mRNA expression of NANOG and MSI1 genes. TWIST1 gene ectopic expression in KYSE-30 cells enhanced the level of cancer stem cell markers' mRNA expression. These results may emphasize the role of TWIST1 in the self-renewal process and may corroborate the involvement of TWIST1 in the stemness state capacity of ESCC cell line KYSE-30, as well as its potential as a therapeutic target.