Tumor Epithelial-mesenchymal Transition Research Articles

Abstract 5304: A promising combination partner for paclitaxel in triple-negative breast cancer (TNBC) therapy

Abstract Cytotoxic chemotherapy is initially effective in patients with metastatic triple-negative breast cancer (mTNBC). However, most of the TNBC patients have high risk of recurrence after chemotherapy and metastatic tumor recurrence indicates rapid tumor proliferation and drug resistance. In TNBC cells, treatment with the chemotherapeutic drug paclitaxel (PTX) induces Transforming growth factor (TGF)-β signaling pathway. TGF-β is a master regulator of tumor angiogenesis and epithelial-mesenchymal transition (EMT), which are limiting the efficacy of cancer therapies and suppressing the anti-tumor immune response. TGF-β induces autophagy in tumor cells and autophagy is closely associated with cancer cell survival and contributing to resistance to therapies. The targeting of TGF-β pathway is possibly an effective strategy to inhibit drug resistance and increase the sensitivity of cancer treatment. Charis 1000 (C1K), which is currently under development, is a synthetic peptide that targets TGF-β1. As C1K partially inhibits TGF-β1-induced Smad2 phosphorylation in TNBC cells, there is no risk of side effects unlike small molecules or antibodies that completely inhibit the TGF-β1 signaling pathway. Combination therapies including chemotherapy are actively investigated as effective treatments for various cancers. PTX, one of frequently used drugs in combination therapies, increases phosphorylation of Smad2, a TGF- β1 downstream signaling pathway and induces autophagy in TNBC cells, but PTX in combination with C1K increases apoptosis while reducing Smad2 phosphorylation and autophagy. The combination of C1K with PTX in the mouse TNBC model showed significant tumor size reduction in comparison to the PTX alone group. In addition, the combination of C1K with PTX confirmed the increased apoptosis and decreased autophagy in the tumor tissues. Furthermore, the combination of C1K with PTX increased the survival rate of mice that was reduced due to the side effects of PTX. These data indicate that the drug resistance induced by TGF-β signaling pathway can be suppressed by C1K combination through TGF-β signaling modulation. C1K may be a promising combination partner for cancer treatment by reducing drug resistance and increasing apoptotic potential of anticancer drugs. Currently, a phase1 clinical trial of the C1K combination therapy with PTX is ongoing and the preclinical studies of the C1K combination with immune checkpoint inhibitors (ICIs) are actively investigated as well. Citation Format: Youl-Nam Lee, Yun-hee Han, Won-Sam Kim, Cheol-Min Lee, Eun-Joung Moon, Hae-Jin Kim. A promising combination partner for paclitaxel in triple-negative breast cancer (TNBC) therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5304.

Safety, pharmacodynamic, and clinical response evaluation of nilotinib and paclitaxel in adults with refractory solid tumors.

3026 Background: The combination of the BCR-Abl kinase inhibitor nilotinib and the anti-tubulin agent paclitaxel was identified in the NCI-ALMANAC study to have greater-than-additive activity in the NCI-60 cell line panel and greater-than-single-agent antitumor activity in xenograft models, in which this combination induces tumor epithelial-mesenchymal transition (EMT). A phase 1 study was initiated to establish the safety, tolerability, and recommended phase 2 dose (RP2D) of this combination in patients (pts) with advanced solid tumors and to examine the pharmacokinetic (PK) and pharmacodynamic (PD) effects of the combination to understand the mechanism of action (NCT02379416). The dose escalation phase established the RP2D as 300 mg oral nilotinib twice daily and 80 mg/m2 intravenous paclitaxel on days (D) 1, 8, and 15 of each 28-day cycle. Here, we report the safety, preliminary PD, and efficacy data for this combination. Methods: Nilotinib and paclitaxel were administered as noted above, with a 1-day (escalation cohort) or 2-day (expansion cohort) paclitaxel-only run-in during the first cycle to enable comparison of the PK and PD effects of the combination vs. single-agent paclitaxel. Paired biopsies to assess tumor molecular response were collected from expansion cohort pts at baseline, cycle (C) 1 D2, and C1D28, with an optional biopsy at progression; accrual continued until ≥ 12 sufficient-quality paired biopsies were obtained. Blood specimens to assess molecular responses in circulating tumor cells (CTCs) were obtained at several timepoints during C1 and longitudinally every cycle thereafter. EMT biomarkers were measured in tumor and CTC specimens using quantitative immunofluorescence microscopy assays. Results: A total of 44 pts were enrolled. Three pts had partial responses (PR), and 1 had an unconfirmed PR (9%); 23 pts (52%) had a best response of stable disease (SD), including 7 pts on study for ≥ 10 cycles. The most common grade (Gr) 3-4 treatment-related adverse events were hematologic and hypophosphatemia. No pts experienced Gr ≥ 3 peripheral neuropathy. The median time on treatment was 67 days. Two pts with granulosa cell ovarian carcinoma had durable responses, completing 74+ and 64 cycles. Multiple patient biopsies and corresponding CTC specimens exhibited treatment-induced EMT. Longitudinal analysis of CTC EMT phenotypes in the 2 pts with extended PR revealed a substantial increase in mesenchymal-like CTCs prior to progression for the pt on study for 64 cycles; such increases were not observed in the pt still on study after 74+ cycles. Further PD analyses are ongoing. Conclusions: The combination of nilotinib and paclitaxel demonstrates promising disease control with durable response in select patients. Tumor PD analyses to discover the underlying pharmacology of this active regimen are ongoing. Funded by NCI Contract No. HHSN261201500003I. Clinical trial information: NCT02379416.

FAM96A suppresses epithelial–mesenchymal transition and tumor metastasis by inhibiting TGFβ1 signals

Metastasis is the main cause of death in 90% of patients with tumors, and epithelial-mesenchymal transition (EMT) plays a key role in this process. Family with sequence similarity 96 member A (FAM96A) is an evolutionarily conserved protein related to cytosolic iron assembly. However, no research has been conducted on its role in tumor metastasis. Bioinformatics analysis with Kaplan–Meier analysis showed that patients with lower FAM96A expression in different tumors had shorter survival times and poorer prognoses. Here, we identified FAM96A as a crucial regulator of the TGFβ signaling pathway because it inhibits TGFβ-mediated tumor metastasis and EMT. FAM96A did not affect the proliferation or apoptosis of tumor cells but significantly reduced their migration and invasion abilities in vitro. The colonization and metastatic abilities of FAM96A-knockout tumor cells were significantly enhanced in vivo. Furthermore, the overexpression of exogenous FAM96A inhibited TGFβ-induced EMT through the SMAD-mediated pathway and downregulated the expression of endogenous transforming growth factor-β1 (TGFβ1). These findings demonstrated a correlation between EMT and FAM96A gene expression for the first time, which is highly important for revealing the mechanism underlying tumor metastasis.

TFEB and TFE3 Depletion Increases Cancer Stem Cell Characteristics and EMT in Triple‐Negative Breast Cancer

Breast cancer is currently the second leading cause of death for women worldwide. One subtype of breast cancer with poor prognosis is triple‐negative breast cancer (TNBC), which is characterized by the lack of expression of estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. TNBC is difficult to treat due to its drug resistance and aggressiveness. Tumor invasiveness is driven by epithelial‐mesenchymal transition (EMT), which allows cells to acquire mobility to metastasize. EMT is also relevant to maintaining cancer stem cell (CSC) characteristics. Drug resistance is driven by CSCs, which are slow to divide, thus surviving chemotherapies, the standard of treatment. Thus, new treatments that inhibit CSCs and/or EMT in TNBC are highly desirable. TFEB and TFE3, two transcription factors of the MiT‐TFE family that operate as a complex, are involved in regulating lysosome biogenesis and autophagy. Our preliminary data and other studies have shown that TFEB and TFE3 regulate pluripotency and EMT in various in vitro models such as gastric and pancreatic cancer, but this regulation is poorly understood in TNBC. To develop new targeted therapies for TNBC, this study aimed to investigate the effects of depleting TFEB and TFE3 on CSC abundance and EMT. CSC renewal capacity was assessed via mammosphere assays and flow cytometry using CD44 and ALDH1A. The MDA MB 231 TFEBKO cells grew the highest number and largest mammospheres compared to the control, TFE3 knock‐down, and double knock‐out cell lines. TFEBKO and TFEBKOTFE3sh showed increases in CD44 expression and ALDH1A activity. While ALDH1A positivity was 1.16% in controls, TFEBKO and TFEBKOTFE3sh significantly increased the population to 22.6% and 21.9%, respectively. Western blotting and a wound healing assay were used to evaluate EMT marker expression and migration, respectively. Quantification revealed that TFEBKO had a two‐fold increase in protein expression levels of EMT markers Vimentin, Snail, and Slug, while TFE3sh showed higher protein expression levels of Vimentin and Slug. TFEBKOTFE3sh showed higher protein expression levels of Slug. The TFEBKO cell line had the highest rate of migration when compared to the control. In conclusion, the data showed that knockout of TFEB and combined knockout of TFEB and TFE3 resulted in an increase in stem cell character and EMT, which could correlate to increased tumor metastasis and poorer prognosis. These results are promising to investigate how depletion or inhibition of TFEB and TFE3 can affect TNBC tumor cell stem cell character and EMT.

A neutrophil-mediated carrier regulates tumor stemness by inhibiting autophagy to prevent postoperative triple-negative breast cancer recurrence and metastasis

Recurrence and metastasis after resection are still the main challenges in clinical treatment of breast cancer. Residual tumor and cancer stem-like cells are the primary culprits of recurrence and metastasis. Recent research studies indicate that autophagy is a cytoprotective mechanism of tumors, which maintains the stemness of cancer cells and promotes tumor proliferation and metastasis. Here, we constructed a “Trojan horse” using neutrophils as the carrier (PH-RL@NEs) to prevent the recurrence and metastasis of postoperative breast cancer. Neutrophils, as a “Trojan horse,” can quickly respond to postoperative inflammation and accurately deliver drugs to the residual tumor site. The inflammation-triggered “Trojan horse” was then opened to release the liposomes containing the chemotherapeutic drug paclitaxel (PTX) and the autophagy inhibitor hydroxychloroquine (HCQ). We found that HCQ could effectively inhibit tumor cell autophagy, interfere with tumor epithelial-mesenchymal transition, and reduce the tumor stem cell-like population. In the orthotopic 4T1 postoperative recurrence models, PTX and HCQ synergistically killed tumors and regulated the stemness of tumor cells, thereby significantly inhibiting tumor recurrence and metastasis. Our work proved that the inhibition of autophagy to reduce tumor stemness is feasible and effective, which opens up a new prospect for postoperative tumor treatment. Statement of significanceThe present study aimed to solve the issues of postoperative recurrence and metastasis of breast cancer and low efficiency of drug administration after surgery. For this purpose, we constructed neutrophils containing hydroxychloroquine (HCQ) and paclitaxel (PTX) co-loaded liposomes (PH-RL@NEs), which for the first time regulated the stemness of tumor cells by inhibiting autophagy, thereby inhibiting postoperative recurrence and metastasis of breast cancer cells. The results showed that PH-RL@NEs enhanced the targeted drug delivery efficiency, with the help of postoperative inflammation chemotaxis of neutrophils. HCQ effectively inhibited autophagy of tumor cells and reduced tumor stem cell-like cells, thus improving the therapeutic effect in the 4T1 in situ postoperative recurrence model.

LncRNA LEF1-AS1 Acts as a Novel Biomarker and Promotes Hypopharyngeal Squamous Cell Carcinoma Progression and Metastasis by Targeting the miR-221-5p/GJA1 Axis.

Hypopharyngeal squamous cell carcinoma (HSCC) is highly malignant and extremely aggressive, making it one of the worst prognoses among all kinds of head and neck squamous cell carcinoma (HNSCC); therefore, gaining insight into molecular mechanisms of HSCC is of profound significance. In the current manuscript, we revealed the elevated expression of long noncoding RNA (lncRNA) LEF1-AS1 in HNSCC which was associated with the poor prognosis by bioinformatic analysis. Moreover, we noticed that LEF1-AS1 dramatically accelerated the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process in HSCC cell line FaDu. Most importantly, we illustrated that LEF1-AS1 played as a competitive endogenous RNA (ceRNA) via sponging miR-221-5p and thereby positively regulated gap junction protein alpha 1 (GJA1) expression, thus aggravated tumor progression and EMT. In conclusion, for the first time, we demonstrated lncRNA LEF1-AS1 as a novel biomarker for HNSCC and suggested LEF1-AS1/miR-221-5p/GJA1 axis as promising diagnostic and therapeutic target for HSCC treatment.

Cinobufotalin inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells through down-regulate β-catenin in vitro and in vivo

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality. The prognosis of HCC is poor due to the high postoperative recurrence rate and metastasis rate. Epithelial-mesenchymal transition (EMT) plays a key role in the metastasis of HCC, which is closely related to the invasion, intrahepatic metastasis and low survival rate. Here we demonstrated that cinobufotalin can upregulate epithelial markers (E-cadherin) and downregulate mesenchymal markers (N-cadherin, snail, slug and ZEB1) in HepG2, SMMC-7721 and SNU-368 cells. We further found that the mRNA and protein expression of β-catenin and its target genes (i.e. MMP7 and DKK1), which are related to tumor invasion and metastasis, were decreased after cinobufotalin treatment. Overexpression of β-catenin promoted EMT of HepG2 and SMMC-7721 cells, and cinobufotalin could antagonize this induction. While Knockdown of β-catenin could inhibit EMT and cinobufotalin enhanced this inhibition. In addition, cinobufotalin significantly suppressed the tumor EMT, as demonstrated by increased E-cadherin expression and decreased N-cadherin and vimentin expression, and inhibited formation and metastasis of lung metastases in vivo. In conclusion, our study has revealed a novel anticancer mechanism of cinobufotalin, which inhibits EMT progress by downregulating β-catenin, and then prevents the migration and invasion of HCC. These results provide convincing evidence for the development of cinobufotalin as a potential HCC metastasis inhibitor.

MicroRNA-124 inhibits canine mammary carcinoma cell proliferation, migration and invasion by targeting CDH2

Canine mammary carcinoma (CMC) is the most common malignant tumor and the second leading cause of cancer-related mortality of dogs worldwide. MicroRNA-124 (miR-124) is an important tumor suppressor implicated in various aspects of carcinogenesis. However, the roles and mechanisms of miR-124 in CMC development remains to be determined. We used the quantitative real-time polymerase chain reaction (qRT-PCR) assay to evaluate the expression levels of miR-124 in CMC tissues obtained from 20 CMC cases and CHMm and CHMp cells. CMC cell lines were transfected with lipfactormine™2000, and the cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Transwell assay were employed for evaluating the cell invasion and migration, while western blot assay was used to detect the protein changes in epithelial-mesenchymal transition (EMT) and CDH2 protein levels. The relationship between miR-124 and the 3′-untranslated region (3'-UTR) of CDH2 was predicted via bioinformatics analysis and verified by dual-luciferase reporter assay. The results revealed that miR-124 was reduced in CMC tissues and cell lines. Besides, observed high histological grade and tumor metastasis were associated with the down-regulation of miR-124 and up-regulation of CDH2. Functional analyses showed that in vitro transfection of CHMm and CHMp cells with miR-124 mimics inhibited their proliferation, migration, invasion, and EMT; however, transfection with miR-124 inhibitor resulted in the reversed effect. Besides, we showed that miR-124 directly suppressed the expression of CDH2, leading to the inhibition of CHMm cell proliferation and EMT. In conclusion, miR-124 regulates CMC tumor growth and EMT by targeting CDH2, maybe a potential therapeutic strategy against CMC.

Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGF\u03b2R2 signaling pathway

BRCA1 deficient breast cancers are aggressive and chemoresistant due, in part, to their enrichment of cancer stem cells that can be generated from carcinoma cells by an epithelial-mesenchymal transition (EMT). We previously discovered that BRCA1 deficiency activates EMT in mammary tumorigenesis. How BRCA1 controls EMT and how to effectively target BRCA1-deficient cancers remain elusive. We analyzed murine and human tumors and identified a role for Tgfβr2 in governing the molecular aspects of EMT that occur with Brca1 loss. We utilized CRISPR to delete Tgfβr2 and specific inhibitors to block Tgfβr2 activity and followed up with the molecular analysis of assays for tumor growth and metastasis. We discovered that heterozygous germline deletion, or epithelia-specific deletion of Brca1 in mice, activates Tgfβr2 signaling pathways in mammary tumors. BRCA1 depletion promotes TGFβ-mediated EMT activation in cancer cells. BRCA1 binds to the TGFβR2 locus to repress its transcription. Targeted deletion or pharmaceutical inhibition of Tgfβr2 in Brca1-deficient tumor cells reduces EMT and suppresses tumorigenesis and metastasis. BRCA1 and TGFβR2 expression levels are inversely related in human breast cancers. This study reveals for the first time that a targetable TGFβR signaling pathway is directly activated by BRCA1-deficiency in the induction of EMT in breast cancer progression.

Quantifying the Epithelial-to-Mesenchymal Transition (EMT) from Bench to Bedside

Simple SummaryCell developmental programs used in wound healing and development such as the epithelial-to-mesenchymal transition (EMT) are frequently coopted by solid tumors to increase motility, plasticity, and invasive characteristics which promote metastasis. Identifying and quantifying the presence and extent of these programs can help to aid in patient prognosis and dictate therapeutic decision making. Here, we review the methods and findings to detect and quantify these cellular transitions in both laboratory and clinical settings.The epithelial-to-mesenchymal transition (EMT) and its reversal, the mesenchymal-to-epithelial transition (MET) are critical components of the metastatic cascade in breast cancer and many other solid tumor types. Recent work has uncovered the presence of a variety of states encompassed within the EMT spectrum, each of which may play unique roles or work collectively to impact tumor progression. However, defining EMT status is not routinely carried out to determine patient prognosis or dictate therapeutic decision-making in the clinic. Identifying and quantifying the presence of various EMT states within a tumor is a critical first step to scoring patient tumors to aid in determining prognosis. Here, we review the major strides taken towards translating our understanding of EMT biology from bench to bedside. We review previously used approaches including basic immunofluorescence staining, flow cytometry, single-cell sequencing, and multiplexed tumor mapping. Future studies will benefit from the consideration of multiple methods and combinations of markers in designing a diagnostic tool for detecting and measuring EMT in patient tumors.

Abstract P3-06-01: Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression and sensitizing breast tumors to subsequent chemotherapy

Abstract Introduction: Two major hurdles faced in clinical cancer therapy are metastatic progression that results in the majority of cancer deaths, and resistance to therapy that fuels tumor relapse. The epithelial-mesenchymal transition (EMT) is a reversible cellular program that contributes to the intratumoral heterogeneity of carcinoma cells and confers traits that promote metastatic progression and resistance to chemotherapy. The standard of care for triple negative breast cancer (TNBC) remains neoadjuvant chemotherapy where cyclophosphamide, doxorubicin and paclitaxel are administered in combination. In case of advanced/metastatic disease, third-line eribulin is administered as a single agent. In some cases of advanced disease, vinorelbine has also demonstrated efficacy. Eribulin, vinorelbine, and paclitaxel are microtubule inhibitors that act via mitotic blockade. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in cancer therapy have been minimal and yielded no clinically viable strategies to selectively modulate EMT in human tumors. Results: Our new data reveals that eribulin, in addition to blocking tubulin polymerization, utilizes microtubule-independent mechanism to epigenetically induce MET. Using thermal proteome profiling and mass spectrometry analyses, we identify candidate target proteins of eribulin that enable it to act in an epigenetic fashion to induce MET.Treating TNBC patient-derived xenograft tumors with eribulin induces a significant reduction in metastatic burden, with the remaining metastatic colonies exhibiting a well-differentiated histopathology. Eribulin efficiently induce MET in cells that have received no prior chemotherapy; in cells pretreated with anthracyclines and taxanes, eribulin is less efficient at MET induction. Additionally, cells that were resistant to first-line taxane treatment were also resistant to eribulin-induced cell death. Using patient-derived xenograft (PDX) models, we have determined the optimal sequence of eribulin administration to maximize its MET-inducing and cytotoxic effects. Conclusions: We uncover that, working through altering the chromatin landscape and transcriptional profile of cells, eribulin induces MET and primes cells for subsequent chemotherapy. Additionally, the efficacy of eribulin is highest when administered in the treatment-naïve setting. By developing an understanding of eribulin’s mechanism of action, treatment strategies can be optimized to maximize therapeutic potential by exploiting its effects in both mitotic blockade as well as MET. Upon completion of this study, we will better understand how MET-induced tumor differentiation would work and the impact that it would have on current conventional therapeutic strategies. Acknowledgements: Eribulin studies funded by Eisai Inc. Citation Format: Diwakar R Pattabiraman, Meisam Bagheri, Nevena B Ognjenovic, Ian S LaCroix, Scott A Gerber. Eribulin alters the chromatin landscape to induce MET, attenuating metastatic progression and sensitizing breast tumors to subsequent chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-01.

Abstract P4-07-19: Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state

Abstract Background: Triple Negative Breast Cancer (TNBC) is an aggressive and heterogeneous subtype characterized by ER/PR/HER2 negative status. Much of the disease potential and aggressive nature of this subtype derives from inter- and intra-tumoral heterogeneity, which makes developing targeted therapies challenging. A key contributor to both heterogeneity in TNBC and later stage chemo-resistance and metastasis is the Epithelial-to-Mesenchymal transition (EMT). This developmental program is frequently exploited in the context of cancer to increase migratory abilities, invasiveness, metastatic potential, and resistance to chemotherapy. Indeed, EMT has been demonstrated and linked to poor prognosis and decreased survival in many solid cancer types. Cells have been found to reside in multiple stable intermediate states along the EMT spectrum, which confer increased aggressive, metastatic, and chemoresistance attributes to a heterogeneous tumor through increased stem-like characteristics. Identifying and targeting this disease-potentiating population in patient tumors is a major hurdle in overcoming metastatic disease. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in diagnostic pathology or cancer therapy have been minimal. One reason for this gap stems from our inability to accurately detect and quantify epithelial-mesenchymal heterogeneity in primary tumor specimens. Secondly, the significance of recently identified intermediate or partial EMT states to predicting tumor prognosis and therapy response are unclear. Approach & Results: To study the role of various states within the EMT spectrum and their regulatory networks, the heterogeneous breast cancer cell line, SUM149PT, was used to derive six single cell clones encompassing the spectrum of EMT states, from epithelial to mesenchymal. Interrogation of this model system in vivo has revealed increased tumor growth and metastatic potential in the intermediate EMT states when compared to the extreme epithelial and mesenchymal states. To further elucidate EMT states in vivo, we employ a 6-marker multi-round immunofluorescence-based staining approach to identify cells that reside in various states along the EMT spectrum. We subsequently used an entropy-based approach and nearest-neighbor analysis on these tumors with the purpose of scoring heterogeneity and overall EMT state. Notably, this analysis segregated stromal infiltrates and their contributions to aggregate EMT scoring, which has been a major hurdle in using EMT as a scoring metric in patient samples. Overall, SUM149 clone-derived tumors held true to the relative EMT states of the starting cell populations; intermediate-derived tumors displayed high heterogeneity while epithelial and mesenchymal clone-derived tumors had lower levels of heterogeneity, despite retaining different EMT scores. Decoupling of heterogeneity and EMT state in this way provides two metrics to assess potential metastatic ability of a tumor. This staining method and analysis has been successfully applied in a preliminary set of patient tumors, showing promise for these two factors, E-M Heterogeneity and EMT score, as a tumor prognostic indicator to inform therapeutic decision-making. Conclusions: EMT tumor states and EMT-derived intra-tumoral heterogeneity play an important role in tumor metastasis and disease progression. Here, we have validated a multiplexed staining approach to quantify these metrics within a tumor, while segregating out stromal infiltrating cells. In the future, this staining and quantification shows promise as a means of predicting patient prognosis and informing potential treatment options based on targeting EMT states Citation Format: Meredith S Brown, Behnaz Abdollahi, Nevena Ognjenovic, Kristen E Muller, Saeed Hassanpour, Diwakar R Pattabiraman. Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-19.

LGR5 expression and clinicopathological features of the invasive front in the fat infiltration area of pancreatic cancer

BackgroundLeucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a strong cancer stem cell marker in colorectal cancer; however, there are many unclear aspects of LGR5 expression in pancreatic cancer. It has been reported that the interaction between tumor cells and stroma at the fat infiltration site has a significant effect on pancreatic cancer prognosis. Therefore, we report a clinicopathological study of LGR5 expression at the fat invasion front in pancreatic cancer.MethodsLGR5 expression was analyzed in 40 pancreatic ductal adenocarcinoma cases with RNAscope, which is a newly developed high-sensitivity in situ hybridization method. Epithelial-mesenchymal transition (EMT) was analyzed by the expression of E-cadherin and vimentin via immunohistochemistry.ResultsLGR5-positive dots were identified in all cases, especially with glandular formation. In the fat invasion front, a high histological grade showed significantly reduced LGR5 expression compared with a low histological grade (p=0.0126). LGR5 expression was significantly higher in the non-EMT phenotype group than in EMT phenotype group (p=0.0003). Additionally, LGR5 expression was significantly lower in cases with high vascular invasion than in those with low vascular invasion (p=0.0244).ConclusionsThese findings suggest that decreased LGR5 expression in the fat invasion front is associated with more aggressive biological behavior in pancreatic ductal adenocarcinoma, with higher tumor grade, EMT phenotype, and higher vascular invasion.

The TCM Prescription Yi-Fei-Jie-Du-Tang Inhibit Invasive Migration and EMT of Lung Cancer Cells by Activating Autophagy.

Yi-Fei-Jie-Du-Tang (YFJDT) is a traditional Chinese medicine formulation. Our previous studies have demonstrated that YFJDT can be used to treat non-small-cell lung cancer (NSCLC), but its protective effect against NSCLC and its mechanisms remain unclear. In the present study, we evaluated the protective effects and potential mechanisms of YFJDT on a tumor-bearing mouse lung cancer model and A549 cell model. Tumor-bearing mice and A549 cells were treated with YFJDT, tumors were measured during the experiment, and tumor tissues and cell supernatants were collected at the end of the experiment to assess the levels of autophagy and epithelial-mesenchymal transition (EMT)-related proteins. The results showed that YFJDT treatment reduced tumor volume and mass, increased the expression of the autophagy marker LC3, and inhibited EMT-related proteins compared with the model group. Cell survival was reduced in the YFJDT-treated groups compared with the model group, and YFJDT also reduced the migration and invasion ability of A549 cells in a dose-dependent manner. Western blotting detected that YFJDT also upregulated FAT4 in the tumor tissue and A549 cells and downregulated the expression of vimentin. Meanwhile, apoptosis in both tissues and cells was greatly increased with treatment of YFJDT. We further interfered with FAT4 expression in cells and found that the inhibitory effect of YFJDT on EMT was reversed, indicating that YFJDT affects EMT by regulating FAT4 expression. Taken together, results of this study suggested that the inhibitory effect of YFJDT on EMT in lung cancer tumors is through upregulating FAT4, promoting autophagy, and thus inhibiting EMT in cancer cells.

MicroRNA-143-3p/TBX3 Axis Represses Malignant Cell Behaviors in Bladder Cancer.

Objective To offer new insight for bladder cancer therapy through researching the microRNA-143-3p/TBX3 axis. Methods Differentially expressed microRNAs in bladder cancer were provided by databases to find microRNA that may regulate TBX3. qRT-PCR was utilized to test levels of TBX3 mRNA and microRNA-143-3p. Their binding was verified with a dual-luciferase method. Malignant cell behaviors were examined by cell functional experiments. Levels of TBX3 protein and proteins pertinent to epithelial-mesenchymal transition (EMT) were tested by western blot. Results TBX3 was highly expressed in bladder cancer cells. MicroRNA-143-3p presented the most conspicuously negative correlation with TBX3, and they had binding sites. Cell functional experiments proved that TBX3 facilitated bladder cancer cell functions and EMT. MicroRNA-143-3p was demonstrated to downregulate TBX3 expression. Rescue assay further illuminated that microRNA-143-3p repressed bladder cancer cell functions and EMT through downregulating TBX3 expression. Conclusion These data all indicated that TBX3 was modulated by microRNA-143-3p and acted as a cancer promoter gene in bladder cancer progression via affecting tumor proliferation, migration, invasion, and EMT. Therefore, a microRNA-143-3p/TBX3 network might be an underlying target for bladder cancer.

RAB4A GTPase regulates epithelial-to-mesenchymal transition by modulating RAC1 activation

Epithelial-to-mesenchymal transition (EMT) is a critical underpinning process for cancer progression, recurrence and resistance to drug treatment. Identification of new regulators of EMT could lead to the development of effective therapies to improve the outcome of advanced cancers. In the current study we discovered, using a variety of in vitro and in vivo approaches, that RAB4A function is essential for EMT and related manifestation of stemness and invasive properties. Consistently, RAB4A suppression abolished the cancer cells’ self-renewal and tumor forming ability. In terms of downstream signaling, we found that RAB4A regulation of EMT is achieved through its control of activation of the RAC1 GTPase. Introducing activated RAC1 efficiently rescued EMT gene expression, invasion and tumor formation suppressed by RAB4A knockdown in both the in vitro and in vivo cancer models. In summary, this study identifies a RAB4A-RAC1 signaling axis as a key regulatory mechanism for the process of EMT and cancer progression and suggests a potential therapeutic approach to controlling these processes.

Predictive Value of Circulating Tumor Cells in Prognosis of Stage III/IV Colorectal Cancer After Oxaliplatin-based First-line Chemotherapy.

Insufficient data exist to support the concept of the circulating tumor cell (CTC) level as a prognostic factor for platinum-based first-line chemotherapy. This study investigated the impact of CTCs on the prognosis of patients with advanced colorectal cancer (CRC) after receiving platinum-based chemotherapy. Analyses were carried out of clinicopathological features and molecular phenotypes to clarify independent risk factors for a high CTC count. Patients diagnosed with stage III/IV CRC (n=76) were included in the study. The blood samples of patients were evaluated for CTCs using the CellRich™ platform system. Immunohistochemistry (Ias used to analyze epithelial-mesenchymal transition-associated biomarkers E-cadherin and vimentin. Univariate and logistic regression analyses were then conducted to analyze the risk factors for CTC expression. Additionally, the influence of oxaliplatin on disease-free survival after first-line chemotherapy or during chemotherapy was analyzed through a 2-year follow-up. Patients in the CTC+ group experienced shorter DFS after receiving oxaliplatin first-line chemotherapy than patients in the CTC- group (p<0.01). In addition, univariate analysis revealed that the tumor M-stage, tumor location, RAS mutation, high expression of vimentin, and deletion of E-cadherin expression were correlated with a high CTC count. Multivariate analysis suggested that the presence of RAS gene mutations and high vimentin expression were independent risk factors for high CTC loads (p<0.01). CTC positivity can indicate the efficacy of first-line chemotherapy with oxaliplatin in stage III/IV colorectal cancer. This may be linked to tumor epithelial-mesenchymal transition in patients with CTCs. Moreover, RAS gene mutation and high expression of vimentin were identified as independent risk factors for a high CTC count.

SNHG16 upregulation-induced positive feedback loop with YAP1/TEAD1 complex in Colorectal Cancer cell lines facilitates liver metastasis of colorectal cancer by modulating CTCs epithelial-mesenchymal transition.

Circulating tumor cells (CTCs) are important precursors of colorectal cancer (CRC) metastasis. The epithelial-mesenchymal transition (EMT) process facilitates CTC invasion by allowing these cells to evade antimetastatic checkpoints to mediate distant metastasis. However, the specific molecular mechanism of tumor EMT remains largely unknown. Based on our previous research on the YAP1 pathway, we further studied the upstream molecule small nucleolar RNA host gene 16 (SNHG16), whose expression was correlated with advanced TNM stage, distant metastasis, and poor prognosis in CRC patients. Furthermore, loss- and gain-of-function assays revealed that SNHG16 promoted CRC colony formation, proliferation, migration, invasion, EMT, mesenchymal-like CTC generation, and liver metastasis through YAP1. Mechanistically, SNHG16 acted as a miRNA sponge to sequester miR-195-5p on Ago2, thereby protecting YAP1 from repression. Moreover, YAP1 bound TEA domain transcription factor 1 (TEAD1) to form a YAP1/TEAD1 complex, which in turn bound two sites in the promoter of SNHG16 and regulate SNHG16 transcription. Finally, in vivo experiments showed that the inhibition of SNHG16 suppressed tumor progression, and that YAP1 rescued the effect of SNHG16 on tumor progression. Herein, we have clarified a hitherto unexplored SNHG16-YAP1/TEAD1 positive feedback loop, that may be a candidate target for CRC treatment.

Molecular Characterization of Clear Cell Renal Cell Carcinoma Reveals Prognostic Significance of Epithelial-mesenchymal Transition Gene Expression Signature

BackgroundThere is an ongoing need to develop prognostic biomarkers to improve the management of clear cell renal cell carcinoma (ccRCC). ObjectiveTo leverage enriched pathways in ccRCC to improve risk-stratification. Design, setting, and participantsWe retrospectively identified two complementary discovery cohorts of patients with ccRCC who underwent (1) radical nephrectomy (RNx) with inferior vena cava tumor thrombectomy (patients = 5, samples = 24) and (2) RNx for localized disease and developed recurrence versus no recurrence (n = 36). Patients with localized ccRCC (M0) in The Cancer Genome Atlas (TCGA, n = 386) were used for validation. Outcome measurements and statistical analysisA differential expression gene (DEG) analysis was performed on targeted RNA next-generation sequencing data from both discovery cohorts. Using TCGA for validation, Kaplan-Meier survival analysis and multivariable Cox proportional hazard testing were utilized to investigate the prognostic impact of DEGs, cell cycle proliferation (CCP), and a novel epithelial-mesenchymal transition (EMT) score on progression-free (PFS) and disease-specific (DSS) survival. Results and limitationsIn the discovery cohorts, we observed overexpression of WT1 and CCP genes in the tumor thrombus versus the primary tumor, as well as in patients with recurrence versus those without recurrence. A hallmark pathway analysis demonstrated enrichment of the EMT- and CCP-related pathways in patients with high WT1 expression in the TCGA (validation) ccRCC cohort. CCP and EMT scores were derived in the validation cohort, which was stratified into four risk groups using Youden Index cut points: CCPlow/EMTlow, CCPlow/EMThigh, CCPhigh/EMTlow, and CCPhigh/EMThigh. The CCPhigh/EMThigh risk group was associated with the worst PFS and DSS (both p < 0.001). In a multivariable analysis, CCPhigh/EMThigh was independently associated with poor PFS and DSS (hazard ratio = 4.6 and 10.3, respectively; p < 0.001). ConclusionsWe demonstrate the synergistic prognostic impact of EMT in tumors with a high CCP score. Our novel EMT score has the potential to improve risk stratification and provide potential novel therapeutic targets. Patient summaryGenes involved in epithelial-mesenchymal transition provides important prognostic information for patients with clear cell renal cell carcinoma.

Understanding the Complex Milieu of Epithelial-Mesenchymal Transition in Cancer Metastasis: New Insight Into the Roles of Transcription Factors.

Epithelial-mesenchymal transition (EMT) is a physiological program during which polarised, immobile epithelial cells lose connection with their neighbours and are converted to migratory mesenchymal phenotype. Mechanistically, EMT occurs via a series of genetic and cellular events leading to the repression of epithelial-associated markers and upregulation of mesenchymal-associated markers. EMT is very crucial for many biological processes such as embryogenesis and ontogenesis during human development, and again it plays a significant role in wound healing during a programmed replacement of the damaged tissues. However, this process is often hijacked in pathological conditions such as tumour metastasis, which constitutes the most significant drawback in the fight against cancer, accounting for about 90% of cancer-associated mortality globally. Worse still, metastatic tumours are not only challenging to treat with the available conventional radiotherapy and surgical interventions but also resistant to several cytotoxic agents during treatment, owing to their anatomically diffuse localisation in the body system. As the quest to find an effective method of addressing metastasis in cancer intervention heightens, understanding the molecular interplay involving the signalling pathways, downstream effectors, and their interactions with the EMT would be an important requisite while the challenges of metastasis continue to punctuate. Unfortunately, the molecular underpinnings that govern this process remain to be completely illuminated. However, it is becoming increasingly clear that EMT, which initiates every episode of metastasis, significantly requires some master regulators called EMT transcription factors (EMT-TFs). Thus, this review critically examines the roles of TFs as drivers of molecular rewiring that lead to tumour initiation, progression, EMT, metastasis, and colonisation. In addition, it discusses the interaction of various signalling molecules and effector proteins with these factors. It also provides insight into promising therapeutic targets that may inhibit the metastatic process to overcome the limitation of “undruggable” cancer targets in therapeutic design and upturn the current spate of drug resistance. More so, it extends the discussion from the basic understanding of the EMT binary switch model, and ultimately unveiling the E/M cellular plasticity along a phenotypic spectrum via multiple trans-differentiations. It wraps up on how this knowledge update shapes the diagnostic and clinical approaches that may demand a potential shift in investigative paradigm using novel technologies such as single-cell analyses to improve overall patient survival.