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Research ArticleCell biologyOncology
Open Access | 
10.1172/jci.insight.192113
1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
Find articles by Monavarian, M. in: PubMed | Google Scholar
1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
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1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
Find articles by Chen, M. in: PubMed | Google Scholar
1Division of Molecular Cellular Pathology, Department of Pathology, O’Neal Cancer Center, Heersink School of Medicine, The University of Alabama, Birmingham, Alabama, USA.
2UAB Biological Data Science Core, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
3Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona, USA.
4Department of Bioengineering, Indian Institute of Science, Bangalore, India.
5Division of Malignant Hematology and Medical Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA.
7Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.
8Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.
Address correspondence to: Karthikeyan Mythreye, Department of Pathology, O’Neal Comprehensive Cancer Center, UAB, WTI 320B, 1824 Sixth Avenue South, Birmingham, Alabama 35294, USA. Email: mythreye@uab.edu.
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
Find articles by Mythreye, K. in: PubMed | Google Scholar
Authorship note: MM and RR contributed equally to this work as co–first authors. MKJ, LI, EW, AS, IBR, EVB, and MC contributed equally to this work.
Published January 15, 2026 - More info
Published in Volume 11, Issue 4 on February 23, 2026
AbstractAnoikis resistance, or evasion of cell death triggered by matrix detachment, is a hallmark of cancer cell survival and metastasis. We showed that repeated exposure to suspension stress followed by recovery under attached conditions leads to development of anoikis resistance. The acquisition of anoikis resistance was associated with enhanced invasion, chemoresistance, and immune evasion in vitro and distant metastasis in vivo. This acquired anoikis resistance was not genetic, persisting for a finite duration without detachment stress, but was sensitive to CDK8/19 mediator kinase inhibition that could also reverse anoikis resistance. Transcriptomic analysis revealed that CDK8/19 kinase inhibition induces bidirectional transcriptional changes in both sensitive and resistant cells, disrupting the balanced reprogramming required for anoikis adaptation and resistance by reversing some resistance-associated pathways and enhancing others. Both anoikis resistance and in vivo metastatic growth of ovarian cancers are sensitive to CDK8/19 inhibition, thereby providing a therapeutic opportunity to both prevent and suppress ovarian cancer metastasis.
Graphical Abstract
Introduction
Metastasis causes most cancer-related deaths, yet metastatic cancers remain largely incurable. A critical step in metastasis is the ability of tumor cells to survive upon detachment from the extracellular matrix and primary tumor site, enabling their dissemination and circulation to distant sites (1–3). Despite our understanding of these metastatic features, their therapeutic targeting remains underdeveloped.
Ovarian cancers (OCs), comprising several subtypes, are among the most devastating of gynecological cancers and archetypal examples of cancers that leverage the metastatic hallmark of anoikis resistance for both transcoelomic/i.p. and distant metastasis (4–8). As malignant ascites accumulates, tumor cells must survive in suspension and evade cell death (9–12). These cells can then colonize peritoneal and mucosal surfaces or return to primary tumor sites (13). The precise mechanisms by which cells acquire such anoikis resistance remain a subject of intense investigation. Suspension culture studies have been used extensively to understand such mechanisms. However, most of them focus on single time points or long-term suspension. Despite these limitations, tumor-intrinsic signals and pathways that change the ability of cells to undergo cell death upon matrix detachment (14–18) have been identified. These anoikis resistance mechanisms include but are not limited to transcriptional upregulation of critical survival genes, repression of pro-apoptotic genes, and transient expression changes in genes associated with antioxidant defense. Changes in the genes and pathways have also been associated with tumor growth and progression in ovarian and other cancers (19–23). Coordinated regulation of specific reprogramming processes such as epithelial-mesenchymal transition (EMT) (24, 25), or cadherin/integrin switching (26), by oncogenic pathways such as Ras/Erk, PI3K/AKT, Rho, MYC, and TGF-β pathways also impacts i.p. OC survival and growth under anchorage independence (27). However, only a few studies (28, 29) directly compare anoikis-sensitive and -resistant models to understand how anoikis resistance can be reached or prevented and the mechanisms underlying this process.
Given the importance of transcriptional changes to overall cancer progression, selective and potent inhibitors of transcription-associated kinases (30) have emerged and are currently being evaluated in the clinic. Among these, CDK8 and CDK19 are of particular interest as they regulate gene expression programs in response to various cellular stresses and have shown promise in preventing resistance in other contexts (31–33). CDK8 and CDK19 are closely related kinases associated with the transcriptional mediator complex that both positively and negatively regulate transcription (34–36), and their inhibition affects different events associated with transcriptional reprogramming, including EMT (24), cell differentiation (37), and gene expression changes in response to various signals and stressors (34, 35). Notably, CDK8/19 inhibitors have reached clinical trials for solid tumors and leukemias (ClinicalTrials.gov NCT03065010, NCT04021368, NCT05052255, NCT05300438), with utility specifically for gynecological cancers under examination (38).
In this study we describe a model system that tests the effects of repeated exposures to detachment stress followed by attached regrowth to mimic potential in vivo scenarios. We delineate the impact of such repeat exposures to detachment stress on the development of anoikis stress in different OC models. Our phenotypic and transcriptomic characterization of such anoikis-resistant cells reveals nongenetic, transcriptional reprogramming, concomitant with a more aggressive phenotype in vitro and in vivo. We further show that both anoikis resistance and i.p. growth of OC can be suppressed by specific inhibition of CDK8/19 mediator kinases, which can also reverse such acquired anoikis resistance. Further, transcriptomic analysis of the effects of CDK8/19 mediator kinase inhibition reveal positive and negative changes to both core and stress-associated transcriptional responses that rebalance the transcriptional response resulting from anoikis resistance. Our findings define what we believe to be a novel therapeutic strategy for counteracting anoikis resistance and metastasis by specific targeting of CDK8/19-regulated transcriptional reprogramming.
ResultsAttachment-detachment cycles confer anoikis-sensitive OC cells with resistance to cell death in suspension. To study how cells develop anoikis resistance, we first screened a panel of tumor cell lines that span commonly used OC cell line models, a pancreatic cancer cell line (PANC1), a prostate cancer cell line (PC3), primary (nonimmortalized) tumor cells from OC patient ascites (EOC15), and 3 nononcogenic immortalized ovarian surface and fallopian tube epithelial cell lines (IOSE144, FT282, and P201 and P210) (20, 23, 39). Anoikis was measured as percentage live cells in suspension relative to the initial plating numbers after plating in poly-HEMA–coated, ultra-low-attachment (ULA) conditions for 24 hours. All lines were plated at identical density in their growth media. Viability ranged from 36.1% for IOSE144 to 125.2% for OVCAR5 (Figure 1A). Models with <100% viability at 24 hours were designated anoikis sensitive (AnS); those with ≥100% were designated intrinsically anoikis resistant (AnR). A subset of AnR lines assessed for up to 72 hours retained resistance (HEYA8, PANC1, TOV21G), whereas OVCA420 increased cell death by 72 hours (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.192113DS1); hence, 72 hours was used for OVCA420 in subsequent experiments. EOC15 maintained in attachment (Supplemental Figure 1B) also exhibited measurable cell death in suspension at 24 hours (Figure 1A).
Figure 1Development of anoikis resistance upon cyclic exposure to matrix detachment stress. (A) Percent live cells after 24 hours in suspension (trypan blue exclusion). n ≥ 3 per cell line. (B) Schematic of cyclic attached growth and suspension culture. (C) Percent live cells in suspension for indicated AnS cells following cyclic attachment-detachment as in B. (n = 3–12 per cell line.) (D) Doubling time of OV90 parental (AnS) and AnR (P7–P9) cells in attached (7 days) or suspension (10 days) culture. (E) Ki67 (red) normalized to DAPI in OV90 AnS and AnR cells after 24 hours in suspension. n = 3 with each trial including quantitation of at least 4 original magnification, 20×, fields. (F) Live/dead staining (Calcein AM green, live cells and ethidium homodimer; red, dead cells) of AnS and AnR cells after 24 hours in suspension (ULA plates); n = 6. (G) Representative images of cleaved caspase-3 Western blot in AnS and AnR cells after 24 hours in suspension; quantitation of cleaved caspase-3 normalized to β-actin shown below (n = 2). (H) Annexin V/PI flow cytometry (left) and quantitation (right) in AnS and AnR cells after 24 hours in suspension; n = 3. (I) Percent live cells in suspension of in vitro–derived (AnS/AnR) and mouse ascites-derived (at endpoint) OV90 and ID8 cells; n = 2–5. Scale bars: 100 μm (E), 200 μm (F). Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by 2-tailed unpaired t test (D and E) or 1-way ANOVA with Tukey’s test (F, H, and I).
We next asked whether AnS cell lines could develop resistance to loss of attachment. To simulate i.p. and distant metastasis where cells undergo detachment stress followed by attached growth (13, 40), we exposed cells to repeated attachment-detachment cycles (Figure 1B). Of AnS lines with <100% viability at first suspension exposure (P1), 7/9 human lines and 1 mouse line showed substantially reduced cell death after sequential cycles (Figure 1C), maintaining acquired AnR through additional rounds. Two lines (HEY, OVCAR3) appeared to become AnR after 3–4 cycles (Supplemental Figure 1C, HEY) but reverted to sensitivity through subsequent cycles.
We assessed if AnR was due to changes in proliferation rate or population-doubling times. No significant differences in doubling time between parental AnS and isogenic AnR cells were observed in either 2D attached or suspension conditions over 10 days (Figure 1D). Parental AnS CAOV3 could not be assessed in suspension because of extensive cell death, but no differences were seen for AnS/AnR pairs in attached growth (Supplemental Figure 1D). Ki67 staining (Figure 1E) also revealed no significant differences between the AnS and AnR cells. In contrast, live/dead staining of OV90 and CAOV3 parental AnS and isogenic AnR cells in suspension (Figure 1F) and cleaved caspase-3 (CC3) analysis after 24 hours in suspension showed reduced cell death in AnR cells as compared with AnS cells (Figure 1G). Flow cytometry for annexin V/PI indicated significantly higher live/dead ratios (2.8-fold for OV90; 1.6-fold for CAOV3) and reduced apoptosis (3.36-fold lower in OV90; 2.59-fold lower in CAOV3) in AnR versus AnS cells (Figure 1H).
To evaluate if in vitro–developed AnR mimicked in vivo AnR, we injected human OV90 cells and mouse ID8 cells i.p. into immunocompromised and immunocompetent mice, respectively. Ascites-derived cells at endpoint, expanded for 1 passage in vitro, showed resistance to suspension comparable to in vitro–adapted AnR cells (Figure 1I). These data suggest that AnR cells adapted to anoikis in vitro show an AnR phenotype similar to in vivo–adapted AnR cells from ascites.
Acquired resistance to cell death in suspension is adaptive and reversible. To determine whether acquired AnR in vitro was due to clonal selection, genetic mutations, or nongenetic mechanisms, we used a modified Luria-Delbrück fluctuation analysis. Recent studies have adapted the classical Luria-Delbrück test to investigate cancer drug resistance by exposing single-cell–derived clones to targeted therapy and analyzing fluctuations in surviving cell numbers to assess reversible switching between drug-sensitive and drug-tolerant states (41–46). We asked if transient switching between cellular states could similarly drive AnR. We assessed survival fluctuations in suspension across single clones from the parental/AnS OV90 population (Figure 2A; n = 60 single clones). Individual clones were expanded for 20 generations before determining live cell viability in suspension induced by detachment stress in poly-HEMA–coated, ULA plates (Figure 2, A and B; P1 survival, gray bars). Clones were also maintained in 2D cultures, and viability of the expanded clonal populations was measured after 3 (P3) and 6 (P6) passages in 2D (Figure 2, A and B). If clones switched between sensitivity (<100% survival) and resistance (≥100%), it would indicate nonheritable resistance. We observed no significant correlation in survival over time among clones (Figure 2C), which had doubling times ranging from 39 to 62 hours (Supplemental Figure 2A; n = 10 random clones), indicating the absence of fixed clonal states. Mean survival fractions (~0.9) and interclonal fluctuations as quantified by the coefficient of variation (CV: 0.25–0.3) were consistent across different passages (CV values for P1 = 0.283 ± 0.05, P3 = 0.289 ± 0.05, and P6 = 0.259 ± 0.05), with observed fluctuations exceeding those of the parental population (CV = 0.11 ± 0.04, obtained from n = 11 biological repeats), where ± denotes the 95% confidence interval of the CV obtained from bootstrapping. If cells responded purely randomly to stress, fluctuations across clones would mirror population noise. However, the higher CV in clones suggests a memory effect or the presence of prestress states influencing detachment stress responses and anoikis resistance.
Figure 2Acquired anoikis resistance represents a transient memory state in the population. (A) Scheme of single clone expansion (OV90) and survival assessment after 24 hours in suspension at indicated passages. (B) Percent survival of individual clones as in A; n = 60 clones. (C) Linear regression of individual clonal survival in suspension at P1 versus P3 (left) or P6 (right). (D) Schematic (left) and percent survival in suspension of AnR OV90 and CAOV3 cells plotted against generations in attached culture (right).
To test this memory effect, we evaluated the stability of the AnR state in OV90 and CAOV3 cells by propagating AnR cells in attached growth for several generations without suspension stress. When rechallenged with suspension stress, OV90 cells regained anoikis sensitivity after 11–14 generations and CAOV3 cells after 8–9 generations, approximating parental population sensitivity levels (Figure 2D). We applied previously developed analytical formulas (43, 45, 46) to predict what levels of fluctuations would be expected from switching between an AnS state (cell death in suspension) and an AnR state (cell survival and proliferation in suspension). If f is the fraction of cells in the resistant state, feγT = 0.9, where T = 24 hr and growth rate
where Td is the cell-doubling time of AnR cells in suspension that is experimentally determined. Thus, given a value of Td, the fraction of resistant cells can be computed from the above equation, and hence a suspension doubling time of 100 hours for OV90 in suspension (Figure 1D) results in f ≈ 0.76. We used equations derived previously to obtain predicted fluctuations (46). Given clonal expansion before the first survival test in suspension and a transient heritability of 10–11 generations for the resistant state (Figure 2D), the model-predicted fluctuations were much less than 0.01 and 30-fold lower than the observed values (0.25–3) for all biologically relevant values of Td (≥38 hours, OV90 2D doubling time; Figure 1D). Thus, reversible switching between these 2 phenotypic states cannot explain the observed clone-to-clone variations in surviving cells.
To uncover mechanisms underlying interclonal fluctuations, we calculated the effective growth rate during the first suspension test using Ln(Nt/N0)/24, where N0 and Nt are cell numbers at the start and end of the 24-hour suspension period. Clones exhibited significant variation in growth rates, averaging –0.004 hr–1 and a CV of 300%. Approximately two-thirds of clones showed negative growth rates (Nt < N0
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