Subset Of Cancer Cells Research Articles

Cells in the polyaneuploid cancer cell (PACC) state have increased metastatic potential

Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.

Long non-coding RNAs in breast cancer stem cells.

Breast cancer, one of the most commonly diagnosed cancers worldwide, is a heterogeneous disease with high rates of recurrence and metastasis that contribute to its high mortality rate. Breast cancer stem cells (BCSCs) are a small but significant subset of heterogeneous breast cancer cells that possess stem cell characteristics such as self-renewal and differentiation abilities that may drive metastasis and recurrence. Long non-coding RNAs (lncRNAs) are a class of RNAs that are longer than 200 nucleotides in length and do not possess protein-coding properties. An increasing number of studies have shown that some lncRNAs are abnormally expressed in BCSCs, and have great biological significance in the occurrence, progression, invasion, and metastasis of various cancers. However, the importance of lncRNAs, as well as the molecular mechanisms that regulate and promote the stemness of BCSCs, are still poorly understood. In the current review, we aim to summarize recent studies that highlight the role of lncRNAs in tumor occurrence and progression through BCSCs. In addition, the utility of lncRNAs as biomarkers of breast cancer progression, and their potential use as therapeutic targets for treatment of breast cancer, will be discussed.

Abstract 5751: Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis

Abstract Background The major genetic driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation in mouse models. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). Consequently, KrasG12D;Pdx1f/f (Pdx1 knockout) mice present with massively accelerated tumor formation. How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods To elucidate mechanisms responsible for the cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq from pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis and correlated to RNA-seq data. Expression of differentially regulated genes was validated by RNAscope and immunohistochemistry staining. The role of identified target genes was studied in pancreatic cancer cell lines. Results Single-cell ATAC-seq proved as a powerful tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. While sole expression of oncogenic Kras lead to reduced accessibility of acinar differentiation genes in acinar cells, these changes were much more prominent in KrasG12D;Pdx1f/f mice, promoting metaplastic conversion. Notably, acinar cells of KrasG12D;Pdx1f/f animals as well as a proportion of metaplastic lesions in both, KrasG12D and KrasG12D;Pdx1f/f mice, showed elevated accessibility and expression of the Ror2 gene. As a receptor protein tyrosine kinase, Ror2 controls essential signaling pathways, such as Ras-MAPK signaling. By analyzing Ror2 knockout mice, we found that the receptor kinase regulates the identity of metaplastic epithelia. Immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens also revealed ROR2 expression in a subset of cancer cells. Knockdown of ROR2 in pancreatic cancer cell lines significantly decreased cell proliferation, while overexpression induced a profound increase in proliferation and in epithelial-to-mesenchymal transition based on the downregulation of multiple epithelial markers and an upregulation of an array of mesenchymal genes. Conclusions Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 leads to vast alterations of acinar cell identity and significantly accelerated transformation. We identified induced expression of the receptor kinase Ror2, which regulates pancreatic cancer initiation and drives pancreatic cancer cell aggressiveness. Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Erick Davis, Jacee Moore, Ivonne Regel, Filip Bednar, Howard Crawford. Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis. [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 5751.

Abstract 486: Characterization of a novel series of highly selective PARP7 inhibitors

Abstract PARP7 is a cellular stress-induced enzyme that adds mono-ADP-ribose groups to a variety of substrate proteins thereby regulating their function. One such substrate is the kinase TBK1 which regulates activity of cGAS-STING and RIG-I nucleic acid sensing pathways. Up-regulation of PARP7 expression in cancers applies a brake to cytosolic nucleic acid sensing and the Type I interferon response. This creates an immunosuppressive tumor microenvironment (TME) leading to faster tumor growth. In addition, inhibition of PARP7 has also been shown to directly arrest growth in a subset of cancer cells via the promotion of a senescence phenotype, inhibition of autophagy and regulation of metabolism. Through the use of structure-based drug design we describe the characterization of potent and selective inhibitors of PARP7. These molecules block viability of a subset of immortalized and primary cancer cells in 2D culture. We have observed potent cGAS-STING independent growth arrest in a number of cancer cell lines harboring a KRAS G12C mutation, and studies combining PARP7 inhibitors with commercially available KRAS G12C inhibitors yield additional in vitro efficacy. Recent studies have shown that inhibitors targeting oncogenic RAS pathway signaling can activate ULK1/2-mediated autophagy as an adaptive treatment resistance mechanism. Hence, our data support further studies investigating the use of PARP7 inhibitors in KRAS-driven cancers either as single agents or in combination with mutant KRAS inhibitors. Our molecules also stimulate robust induction of type I interferon (IFNβ) and downstream chemokines such as CXCL10 in a cGAS-STING pathway dependent manner in a range of cancer cells and 3D organoids. This induction is augmented by combining agonists of cGAS-STING and RIG-I pathways with PARP7 inhibitors. In addition, PARP7 inhibitors work in concert with exogenous DNA-damaging agents such as chemotherapeutics and X-ray radiation to induce the type I interferon response in cancer cells. Overall, these data highlight the opportunity to utilize PARP7 inhibitors in cancers where high genomic instability leads to aberrant cytosolic nucleic acid levels or in concert with exogenous DNA-damaging agents. Our PARP7 inhibitors exhibit highly desirable physico-chemical and in vitro ADME properties coupled with an exceptional in vitro safety profile which translates to best-in-class rodent PK. In a KRAS G12C lung cancer xenograft model (NCI-H1373), twice daily oral administration of an exemplar PARP7 inhibitor drives rapid and full tumor regression in a dose-dependent manner. The anti-tumor effects correlate with robust tumor PK/PD upon measurement of tumor IFNβ and CXCL10. In summary, we describe the characterization of a lead series of highly potent and selective PARP7 inhibitors which demonstrate excellent in vitro ADMET and in vivo PK properties leading to best-in-class anti-tumor efficacy in a KRAS-driven lung cancer xenograft model. Citation Format: Phillip M. Cowley, Barry E. McGuinness, Gillian M. Campbell, Alan Wise. Characterization of a novel series of highly selective PARP7 inhibitors [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 486.

Abstract 2441: Identification and characterization of the role of KRT17 in the stemness regulation of hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) ranks as the fifth most common cancer and the second leading cause of cancer-related mortality worldwide. The overall prognosis of HCC patients is very poor because it is susceptible to recurrence and metastasis. According to the cancer stem cell (CSC) model, there is a small subset of cancer cells within the tumor bulk that is responsible for tumor relapse and metastasis. CSCs represent cells that can self-renew, initiate tumors, and keep resistant to anti-cancer treatment. However, the fate and signatures of CSCs in HCC remain elusive. Our previous in vitro hepatocyte differentiation model successfully mimicked the liver developmental process from the stage of embryonic stem cells, endoderm, liver progenitor cells, and premature hepatocytes, together with one pair of nontumor and tumor tissue from an HCC patient. We found that the expression of keratin 17 (KRT17) reached its peak at the liver progenitor stage and was down-regulated along with liver development, which shared the same expression pattern with keratin 19 (CK19), a well-known HCC stemness-associated gene. Single-cell RNA sequencing in 9 HCC clinical samples from the GEO dataset revealed that KRT17 was expressed in less than 1% of tumor cells. Moreover, KRT17 shared similar expression patterns among hepatocytes, non-stem like HCC and stem like HCC subpopulations with a panel of hepatic stem cell markers and showed reverse trends with a list of hepatic markers. Clinically, KRT17 was highly expressed in cancer tissues compared to normal counterparts and positively correlated with poor overall survival. Functional assays indicated that KRT17 could considerably facilitate the self-renewal, growth, and metastasis properties of HCC both in vitro and in vivo. Also, KRT17 endowed HCC cells with enhanced resistance to sorafenib treatment. Moreover, the expressions of the CSC marker (CD133) and oncofetal drivers (NANOG and KLF4) were significantly upregulated upon KRT17 overexpression. Bioinformatic analysis using gene expression profiles of patients from the TCGA database revealed that development- and differentiation-related biological processes were dramatically enriched in KRT17-high patients. The underlying molecular mechanism of how KRT17 augments HCC stemness still needs further study. Overall, our study uncovered a new connection between KRT17 and HCC stemness and progression. KRT17 might be a novel marker of CSC and a prognostic biomarker for the treatment of HCC patients. Citation Format: Xiaona Fang, Shan Liu, Liuxian Ban, Jiao Huang, Jie Luo, Lanqi Gong, Baifeng Zhang, Beilei Liu, Jinlin Huang, Yuma Yang, Ching Ngar Wong, Qian Yan, Xin-Yuan Guan. Identification and characterization of the role of KRT17 in the stemness regulation of hepatocellular carcinoma [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 2441.

Long noncoding RNA SNHG15: A promising target in human cancers.

As oncogenes or tumor suppressor genes, lncRNAs played an important role in tumorigenesis and the progression of human cancers. The lncRNA SNHG15 has recently been revealed to be dysregulated in malignant tumors, suggesting the aberrant expression of which contributes to clinical features and regulates various oncogenic processes. We have selected extensive literature focused on SNHG15 from electronic databases, including studies relevant to its clinical significance and the critical events in cancer-related processes such as cell proliferation, apoptosis, autophagy, metastasis, and drug resistance. This review summarized the current understanding of SNHG15 in cancer, mainly focusing on the pathological features, known biological functions, and underlying molecular mechanisms. Furthermore, SNHG15 has been well-documented to be an effective diagnostic and prognostic marker for tumors, offering novel therapeutic interventions in specific subsets of cancer cells.

Abstract P3-05-42: Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis

Abstract Introduction. The CUB domain containing protein 1, also known as TRASK (Transmembrane and associated with Src kinases) or CD318, has been suggested as a biomarker for stem cells or stem like cells. The transmembrane CDCP1 protein is known to activate the Src kinase and has recently shown to play a key part in the aggressiveness of breast cancer cells including migration, invasiveness and possibly growth induced by the motogenic Hepatocyte Growth Factor (HGF) and its receptor MET (Kawase et al 2022), a process requires Rho-GEF, PI3K and STAT5. This appears to allow identification of a subset of cancer cells that are aggressive in their biological behaviours. Here, we examined the expression profile of CDCP1, SRC, the HGF/HGF receptor complex and HGF activation regulators in a cohort of breast cancer and attempt to establish if such protein axis is able to identify patients with high risk of poor clinical outcome. Methods. Using an establish breast cancer cohort which is comprised of both normal mammary tissues and breast cancer tissues freshly obtained after surgery, we quantified the gene transcripts of CDCP1/CD318 and examined its clinical and pathological relevance. Integrated analysis was conducted for CDCP1 and the expression profile of SRC, PI3K, HGF, the HGF receptor cMET, HGF activator (HGFA), HGF activation regulators including HAI-1, HAI2, matriptase-1 and matriptase-2, and STAT family members. This was done against the clinical outcome of the patients as well as the clinical and pathological factors. Results. Breast cancer tissues expressed high levels of CDCP1 compared with normal mammary tissues. CDCP1 itself had a weak yet significant value in predicting the overall survival of the patients (p=0.047). The expression levels of these CDCP1 related molecules aren’t significantly correlated with CDCP1. However, integrated analyses revealed that CDCP1, together with its pathway regulators SRC, HGF, the HGF receptor (MET) and the HGF activation regulators matriptases form a power prognostic indicator for the clinical outcome of the patients (p< 0.0001, HR=1.8 for overall survival (OS) and p=0.002, HR=1.5 for disease-free survival (DFS)). This integrated profile has also identified subgroups of patients with highly favourable and very poor clinical outcomes over a ten-year follow-up period, with the respective survival at 100% and 36% respectively. The predictive power for OS and DFS is highly independent of other clinical and pathological factors in a multivariate analysis (p=0.003 and p=0.017 respectively). The predictive power is applicable to ER negative and HER2 positive tumours. Discussion. CDCP1/CD318, a factor known to stimulate cancer cell aggressiveness, together with its pathway kinase SRC and newly identified extracellular activator HGF and the HGF regulators forms a significant independent prognostic factor. It identifies subgroups of patients with favourable and poor prognosis, allowing consideration of targeted therapies for the patients. Reference Kawase N, Sugihara A, Kajiwara K, Hiroshima M, Akamatsu K, Nada S, Matsumoto K, Ueda M, Okada M. SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells. J Biol Chem, 2022, 298:101630. doi: 10.1016/j.jbc.2022.101630 Citation Format: Yiming Yang, Xuefei Dong, Tracey A. Martin, Lin Ye, Jane Lane, Andrew J. Sanders, QingPing Dou, Eleri Davies, Wen G. Jiang. Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-42.

Abstract P6-11-11: Breast cancer stem cell marker, CD24 regulates metabolic reprogramming in triple negative breast cancer

Abstract Breast cancer stem cell (BCSC) is a subset of cancer cells that can dictate the tumor initiation, metastatic progression, and therapeutic resistance in BC. The eradication of this unusual population is emerging as a new paradigm in cancer treatment. Molecularly, CD24 negativity in conjunction with high expression of CD44 is considered a hallmark for the BCSCs. While extensive studies have been performed to delineate the role of CD44 in BCSCs, the regulation of CD24 and its functional role have not been fully understood. In this study, we investigated the regulatory mechanisms responsible for low CD24 expression in BCSCs and their functional relevance in BCSCs. Analysis of DNA from tumor tissues and blood from BC patients as well as BCSCs sorted from BC cell lines suggest that CD24 is epigenetically regulated via DNMT-1/HDAC1-dependent increased methylation of CpG islands in the CD24 proximal promoter region. To understand the role of CD24 in triple-negative BC (TNBC), an aggressive subgroup of BC, we knocked down (CD24-KD) and overexpressed (CD24-OE) CD24 in metastatic TNBC cells. While CD24-KD resulted in increased proliferation and stemness, CD24-OE diminished the proliferative and stem-like potential. To further identify the signaling cascade underpinning these effects, we performed phospho-proteome analysis using Reverse Phase Protein Array (RPPA). Remarkably, we observed an enhanced activation of AMPK and NF-kB signaling cascades, and reduced PDGFRβ signaling upon depletion of CD24. As signaling cascades are intricately linked to cellular metabolism, we performed a metabolomic analysis of CD24-KD and CD24-OE cells. Our comprehensive analysis revealed heightened activation of mitochondrial fatty acid β-oxidation (FAO) in CD24-KD and increased glutamine metabolism in CD24-OE cells. In coherence with these findings, we observed significant regulation of genes related to fatty acid metabolism in the TNBC patient cohort expressing low levels of CD24. Consistently, the CD24-KD cells demonstrated increased sensitivity towards FAO inhibitors while CD24-OE cells were more sensitive to glutamine metabolism inhibitors. In vivo studies to further understand the translational significance of this metabolic axis is underway. Taken together, our study demonstrates, for the first time, that CD24 presents a novel metabolic vulnerability that can target BCSCs to gain a therapeutic advantage in the treatment of drug-resistant TNBC patients. Citation Format: Divya Murthy, Debasmita Dutta, Sukjin Yang, Junhyoung Park, Benny Kaipparettu. Breast cancer stem cell marker, CD24 regulates metabolic reprogramming in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-11.

CXCR1: A Cancer Stem Cell Marker and Therapeutic Target in Solid Tumors.

Therapy resistance is a significant concern while treating malignant disease. Accumulating evidence suggests that a subset of cancer cells potentiates tumor survival, therapy resistance, and relapse. Several different pathways regulate these purported cancer stem cells (CSCs). Evidence shows that the inflammatory tumor microenvironment plays a crucial role in maintaining the cancer stem cell pool. Typically, in the case of the tumor microenvironment, inflammatory pathways can be utilized by the tumor to aid in tumor progression; one such pathway is the CXCR1/2 pathway. The CXCR1 and CXCR2 receptors are intricately related, with CXCR1 binding two ligands that also bind CXCR2. They have the same downstream pathways but potentially separate roles in the tumor microenvironment. CXCR1 is becoming more well known for its role as a cancer stem cell identifier and therapeutic target. This review elucidates the role of the CXCR1 axis as a CSC marker in several solid tumors and discusses the utility of CXCR1 as a therapeutic target.

CD24+CD44+CD54+EpCAM+ gastric cancer stem cells predict tumor progression and metastasis: clinical and experimental evidence

BackgroundGastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Specific and thorough identification of cancer cell subsets with higher tumorigenicity and chemoresistance, such as cancer stem cells (CSCs), could lead to the development of new and promising therapeutic targets. For better CSC identification, a complete or extended surface marker phenotype is needed to provide increased specificity for new cell targeting approaches. Our goal is to identify and characterize a putative extended phenotype for CSCs derived from patients with GC before treatment, as well as to evaluate its clinical value. In addition, we aim to ensure that cells with this phenotype have stemness and self-renewal capabilities.MethodsThis is a cohort study including 127 treatment-naïve patients with GC who attended the Instituto Nacional de Cancerología. Multiparametric flow cytometry analysis was performed to determine the extended phenotype of cells derived from gastric biopsies. The tumorigenic capability of cells identified in patients was assessed in a zebrafish model.ResultsCD24+CD44+CD54+EpCAM+ cells were present in all treatment-naïve patients included, with a median abundance of 1.16% (0.57–1.89%). The percentage of CD24+CD44+CD54+EpCAM+ cells was categorized as high or low using 1.19% as the cutoff for the CD24+CD44+CD54+EpCAM+ cell subset. Additionally, a higher TNM stage correlated with a higher percentage of CD24+CD44+CD54+EpCAM+ cells (Rho coefficient 0.369; p < 0.0001). We also demonstrated that a higher percentage of CD24+CD44+CD54+EpCAM+ cells was positively associated with metastasis. The metastatic potential of these cells was confirmed in a zebrafish model. Ultimately, under our conditions, we conclude that CD24+CD44+CD54+EpCAM+ cells are true gastric cancer stem cells (GCSCs).ConclusionThe CD24+CD44+CD54+EpCAM+ cells present in tissue samples from patients are true GCSCs. This extended phenotype results in better and more specific characterization of these highly tumorigenic cells. The relative quantification of CD24+CD44+CD54+EpCAM+ cells has potential clinical value, as these cells are associated with metastatic disease, making their presence an additional prognostic marker and possibly a target for the design of new antineoplastic treatments in the era of precision oncology. Overall, the extended CD24+CD44+CD54+EpCAM+ phenotype of GCSCs could support their isolation for the study of their stemness mechanisms, leading to the identification of better molecular targets for the development of both new therapeutic approaches such as oncoimmunotherapy and new diagnostic and clinical prognostic strategies for GC.

Abstract PR010: Aging-induced reprogramming of the cell of origin defines lung cancer evolution

Abstract Aging is one of the most important risk factors for cancer development. However, our understanding of how aging impacts cancer evolution remains at an early stage. We investigated the evolution of lung adenocarcinoma (LUAD) in aged vs. young KrasLSL-G12D/+; Trp53flox/flox (KP) mice. Surprisingly, we found that LUAD initiation and progression were significantly impaired with aging. Using functional in vivo and in vitro assays, we demonstrated that the loss of tumor-initiating potential could be attributed to a loss of stemness in the alveolar type 2 (AT2) cells, which are the predominant cell of origin for LUAD. We next explored the biology of the tumors that formed in aged KP mice using single-cell transcriptomics. We discovered a distinct compositional landscape of cancer cell subsets in aged lung tumors, revealing a delay in the molecular progression of the cancers and an aging-specific gene expression signature. Interestingly, key components of this gene signature could be traced back to specific epigenetic alterations in the AT2 cells. Furthermore, the aged mouse LUAD signature was highly predictive of patient age in human LUAD, indicating conservation of the age-associated changes across species. We hypothesized that the aging-associated changes in gene expression during LUAD tumorigenesis may underpin delayed tumor progression in the aged KP mice. To test this, we performed an in vivo genetic screen in the autochthonous KP model and identified genetic dependencies that were specific to LUAD in aged or young mice. Our results indicate that aging-associated epigenetic changes in the cell of origin are imprinted on evolving tumors, which determines tumor initiation potential, alters trajectory of tumor evolution, and leads to age-dependent vulnerabilities. These findings suggest that the biology of tumors is fundamentally altered by aging, raising new possibilities for cancer treatment and prevention in patients of different ages. Citation Format: Xueqian Zhuang, Simon Joost, Qing Wang, Melissa Blum, Selena Ding, Zhuxuan Li, Yingqian Zhan, Richard Koche, Emily Wong, Tuomas Tammela. Aging-induced reprogramming of the cell of origin defines lung cancer evolution [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR010.

A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

Role of microRNA-regulated cancer stem cells in recurrent hepatocellular carcinoma

Among the most common cancers, hepatocellular carcinoma (HCC) has a high rate of tumor recurrence, tumor dormancy, and drug resistance after initial successful chemotherapy or radiotherapy. A small subset of cancer cells, cancer stem cells (CSCs), exhibit stem cell characteristics and are present in various cancers, including HCC. The dysregulation of microRNAs (miRNAs) often accompanies the occurrence and development of HCC. miRNAs can influence tumorigenesis, progression, recurrence, and drug resistance by regulating CSCs properties, which supports their clinical utility in managing and treating HCC. This review summarizes the regulatory effects of miRNAs on CSCs in HCC with a special focus on their impact on HCC recurrence.

The dual FAK-HDAC inhibitor MY-1259 displays potent activities in gastric cancers in vitro and in vivo

Epigenetic regulation and Focal adhesion kinase (FAK) are considered to be two important targets for the development of antitumor drugs. Studies have shown that the combination of FAK and HDAC inhibitors could exhibit synergistic effects in a subset of cancer cells in vitro and in vivo. At present, there are few reports on dual target inhibitors of FAK and HDAC. Here, we first reported a new compound MY-1259 as a dual FAK and HDAC6 inhibitor, which exhibited efficient treatment effects on gastric cancers in vitro and in vivo. MY-1259 exhibited potent inhibitory activities against FAK (IC50 = 132 nM) and HDAC6 (IC50 = 16 nM). Notably, MY-1259 showed selective inhibitory potency on HDAC6 over HDAC1, HDAC2 and HDAC3. In addition, MY-1259 could potently inhibit the proliferative activities of MGC-803 and BGC-823 cells (IC50 = 3.91 and 15.46 nM, respectively, using flow cytometry counting), induce cell apoptosis, and cellular senescence. MY-1259 could effectively down-regulate the levels of Ac-Histone H3 and Ac-α-tubulin, and also inhibit the phosphorylation of FAK at three phosphorylation sites Y397, Y576/577 and Y925, thereby inhibiting the activation of ERK and AKT/mTOR. MY-1259 exhibited more effective antitumor effect in vivo than the HDAC inhibitor SAHA and FAK inhibitor TAE-226 alone or in combination, showing the advantages of FAK/HDAC dual inhibitors in the treatment of gastric cancers. Therefore, the results in this work suggested that inhibition of FAK and HDAC by MY-1259 might represent a promising strategy for the treatment of gastric cancers.

TERRA increases at short telomeres in yeast survivors and regulates survivor associated senescence (SAS).

Cancer cells achieve immortality by employing either homology-directed repair (HDR) or the telomerase enzyme to maintain telomeres. ALT (alternative lengthening of telomeres) refers to the subset of cancer cells that employ HDR. Many ALT features are conserved from yeast to human cells, with the yeast equivalent being referred to as survivors. The non-coding RNA TERRA, and its ability to form RNA-DNA hybrids, has been implicated in ALT/survivor maintenance by promoting HDR. It is not understood which telomeres in ALT/survivors engage in HDR, nor is it clear which telomeres upregulate TERRA. Using yeast survivors as a model for ALT, we demonstrate that HDR only occurs at telomeres when they become critically short. Moreover, TERRA levels steadily increase as telomeres shorten and decrease again following HDR-mediated recombination. We observe that survivors undergo cycles of senescence, in a similar manner to non-survivors following telomerase loss, which we refer to as survivor associated senescence (SAS). Similar to 'normal' senescence, we report that RNA-DNA hybrids slow the rate of SAS, likely through the elongation of critically short telomeres, however decreasing the rate of telomere shortening may contribute to this effect. In summary, TERRA RNA-DNA hybrids regulate telomere dysfunction-induced senescence before and after survivor formation.

The Intricate Interplay between the ZNF217 Oncogene and Epigenetic Processes Shapes Tumor Progression.

The oncogenic transcription factor ZNF217 orchestrates several molecular signaling networks to reprogram integrated circuits governing hallmark capabilities within cancer cells. High levels of ZNF217 expression provide advantages to a specific subset of cancer cells to reprogram tumor progression, drug resistance and cancer cell plasticity. ZNF217 expression level, thus, provides a powerful biomarker of poor prognosis and a predictive biomarker for anticancer therapies. Cancer epigenetic mechanisms are well known to support the acquisition of hallmark characteristics during oncogenesis. However, the complex interactions between ZNF217 and epigenetic processes have been poorly appreciated. Deregulated DNA methylation status at ZNF217 locus or an intricate cross-talk between ZNF217 and noncoding RNA networks could explain aberrant ZNF217 expression levels in a cancer cell context. On the other hand, the ZNF217 protein controls gene expression signatures and molecular signaling for tumor progression by tuning DNA methylation status at key promoters by interfering with noncoding RNAs or by refining the epitranscriptome. Altogether, this review focuses on the recent advances in the understanding of ZNF217 collaboration with epigenetics processes to orchestrate oncogenesis. We also discuss the exciting burgeoning translational medicine and candidate therapeutic strategies emerging from those recent findings connecting ZNF217 to epigenetic deregulation in cancer.

Abstract B021: Claudin-1 expression promotes chemoresistance and cancer stem cell characteristics in colorectal cancer

Abstract Background: Claudins are integral components of tight junction complexes. Beyond just passive cell adhesion molecules, they influence structural dynamics, molecular trafficking, and important cell signaling pathways. Claudins are often dysregulated in cancer, leading to changes in adhesion and metastatic potential. Claudin-1 (Cldn1) is expressed at low levels in normal colon but is highly expressed in colorectal cancer (CRC) and promotes epithelial-mesenchymal transition and metastasis. Cancer stem cells (CSCs) are a small subset of cancer cells that display increased capacity for self-renewal and can undergo symmetric and asymmetric division to reform tumors after treatment. CSCs are resistant to cytotoxic drugs and are thought to be responsible for treatment failure, metastasis, and relapse. Significance of problem: CRC is the second leading cause of cancer-related deaths in the United States with an estimated 151,030 new cases in 2022. Although CRC has an overall relative 5-year survival rate of 64%, prognosis varies dramatically with the stage at diagnosis. Localized CRC can be treated surgically and has a 5-year survival rate of 91%, while the survival rate of metastatic CRC falls to 15%. The primary problem in treating metastatic CRC is a combination of intrinsic and acquired chemoresistance. Therefore, understanding the mechanisms of chemoresistance is vital to developing effective therapies. Hypothesis: Claudin-1 expression in colorectal cancer increases resistance to chemotherapeutic drugs and promotes cancer stem cell characteristics. Experimental design: Bioinformatic analysis was performed on a database of responders and non-responders to standard-of-care CRC chemotherapy. Cldn1 expression was manipulated in CRC cell lines, and assays were performed to evaluate chemoresistance and CSC characteristics. Results: Bioinformatic analysis of a patient dataset of responders and non-responders to chemotherapy revealed that Cldn1 expression was positively correlated with resistance to FOLFOX treatment. It was also significantly correlated with colorectal CSC marker expression including CD44, CD133, and Lgr5. Overexpression of Cldn1 in SW480 cells led to a significant increase in resistance to 5-FU, side population and ALDH-high percentage, colorectal CSC markers, and tumorigenic potential, while knockout of Cldn1 in SW620 and DLD-1 cells led to a decreased response in these assays. Follow-up experiments identified the PDZ-binding domain of Cldn1 as essential for the chemoresistant phenotype. Specifically, both CD44 and its downstream target phospho-AKT are increased with the expression of full-length but not mutated Cldn1. Conclusion: Cldn1 expression induces chemoresistance and cancer stem cell properties in colorectal cancer. Because of this, Cldn1 may serve as a biomarker in CRC for prescribing more aggressive adjuvant therapy or as a molecule for targeted treatment aimed at the resistant tumor cells responsible for treatment failure and recurrence. Citation Format: Mark Primeaux, Saiprasad Gowrikumar, Dhundy Bastola, Punita Dhawan, Amar Singh. Claudin-1 expression promotes chemoresistance and cancer stem cell characteristics in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B021.

ScAB detects multiresolution cell states with clinical significance by integrating single-cell genomics and bulk sequencing data.

Although single-cell sequencing has provided a powerful tool to deconvolute cellular heterogeneity of diseases like cancer, extrapolating clinical significance or identifying clinically-relevant cells remains challenging. Here, we propose a novel computational method scAB, which integrates single-cell genomics data with clinically annotated bulk sequencing data via a knowledge- and graph-guided matrix factorization model. Once combined, scAB provides a coarse- and fine-grain multiresolution perspective of phenotype-associated cell states and prognostic signatures previously not visible by single-cell genomics. We use scAB to enhance live cancer single-cell RNA-seq data, identifying clinically-relevant previously unrecognized cancer and stromal cell subsets whose signatures show a stronger poor-survival association. The identified fine-grain cell subsets are associated with distinct cancer hallmarks and prognosis power. Furthermore, scAB demonstrates its utility as a biomarker identification tool, with the ability to predict immunotherapy, drug responses and survival when applied to melanoma single-cell RNA-seq datasets and glioma single-cell ATAC-seq datasets. Across multiple single-cell and bulk datasets from different cancer types, we also demonstrate the superior performance of scAB in generating prognosis signatures and survival predictions over existing models. Overall, scAB provides an efficient tool for prioritizing clinically-relevant cell subsets and predictive signatures, utilizing large publicly available databases to improve prognosis and treatments.

Abstract B061: Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analysis

Abstract Background: The major genetic driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation in mouse models. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). Consequently, KrasG12D;Pdx1f/f (Pdx1 knockout) mice present with massively accelerated tumor formation. How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods: To elucidate mechanisms responsible for the accelerated cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) from frozen pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis (two and ten weeks after CreERT-induced recombination) and compared to those of KrasG12D and CreERT control animals. Changes in the epigenome were correlated to RNA-seq data. Expression of differentially regulated genes was validated by RNAscope and immunohistochemistry staining. The role of identified target genes was studied in pancreatic cancer cell lines. Results: Single-cell ATAC-seq proved as a very powerful and robust tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. By analyzing tissue of KrasG12D;Pdx1f/f mice at different time points, we captured the complete metaplastic conversion of acinar cells into ADM and different subsets of precancerous lesions. Despite initially appearing phenotypically normal, the chromatin profiles of Pdx1 knockout acinar cells showed a striking similarity to ADM cells. Interestingly, acinar and ADM cells of KrasG12D;Pdx1f/f animals as well as ADM lesions of KrasG12D mice showed elevated accessibility and expression of the Ror2 gene. As a receptor protein tyrosine kinase, Ror2 regulates essential signaling pathways, such as Ras-MAPK signaling. Immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens also revealed Ror2 expression in a subset of neoplasms and cancer cells. Knockdown of Ror2 in pancreatic cancer cell lines with a basal-like subtype significantly decreased cell proliferation and expression of cell cycle regulators, while ectopic overexpression in PDAC lines with a classical differentiation induced a dramatic change in cell identity and massively increased cell proliferation. Conclusions: Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 lead to vast alterations of acinar cell identity and significantly accelerated transformation. We identified de-regulation of the receptor kinase Ror2, which cooperates with KrasG12D to induce and promote pancreatic carcinogenesis and proliferation. Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Erick Davis, Jacee Moore, Tobias Straub, Ivonne Regel, Filip Bednar, Howard Crawford. Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analysis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B061.

Presence of cells in the polyaneuploid cancer cell (PACC) state predicts the risk of recurrence in prostate cancer.

The nonproliferating polyaneuploid cancer cell (PACC) state is associated with therapeutic resistance in cancer. A subset of cancer cells enters the PACC state by polyploidization and acts as cancer stem cells by undergoing depolyploidization and repopulating the tumor cell population after the therapeutic stress is relieved. Our aim was to systematically assess the presence and importance of this entity in men who underwent radical prostatectomy with curative intent to treat their presumed localized prostate cancer (PCa). Men with National Comprehensive Cancer Network intermediate- or high-risk PCa who underwent radical prostatectomy l from 2007 to 2015 and who did not receive neoadjuvant treatment were included. From the cohort of 2159 patients, the analysis focused on a subcohort of 209 patientsand 38 cases. Prostate tissue microarrays (TMAs) were prepared from formalin-fixed, paraffin-embedded blocks of the radical prostatectomy specimens. A total of 2807 tissue samples of matched normal/benign and cancer were arrayed in nineTMA blocks. The presence of PACCs and the number of PACCs on each core were noted. The total number of cells in the PACC state and the total number of cores with PACCs were significantly correlated with increasing Gleason score (p = 0.0004) and increasing Cancer of the Prostate Risk Assessment Postsurgical(CAPRA-S) (p = 0.004), but no other variables. In univariate proportional hazards models of metastasis-free survival, year of surgery, Gleason score (9-10 vs. 7-8), pathology stage, CAPRA-S, total PACCs, and cores positive for PACCs were all statistically significant. The multivariable models with PACCs that gave the best fit included CAPRA-S. Adding either total PACCs or cores positive for PACCs to CAPRA-S both significantly improved model fit compared to CAPRA-S alone. Our findings show that the number of PACCs and the number of cores positive for PACCs are statistically significant prognostic factors for metastasis-free survival, after adjusting for CAPRA-S, in a case-cohort of intermediate- or high-risk men who underwent radical prostatectomy. In addition, despite the small number of men with complete data to evaluate time to metastatic castration-resistant PCa (mCRPC), the total number of PACCs was a statistically significant predictor of mCRPC in univariate analysisand suggested a prognostic effect even after adjusting for CAPRA-S.