CYPD limits HR+ mammary carcinogenesis in mice

CYPD limits HR+ mammary carcinogenesis in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CYPD limits HR + mammary carcinogenesis in mice Lorenzo Galluzzi, Aitziber Buqué, Manuel Beltrán-Visiedo, Ai Sato, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6265353/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jun, 2025 Read the published version in Cell Death Discovery → Version 1 posted 7 You are reading this latest preprint version Abstract Mitochondrial permeability transition (MPT)-driven necrosis and necroptosis are regulated variants of cell death that can drive inflammation or even promote antigen-specific immune responses. In oncological settings, indolent inflammatory reactions have been consistently associated with accelerated disease progression and resistance to treatment. Conversely, adaptive immune responses specific for tumor-associated antigens are generally restraining tumor development and contribute to treatment sensitivity. Here, we harnessed female C57BL/6J mice lacking key regulators of MPT-driven necrosis and necroptosis to investigate whether whole-body defects in these pathways would influence mammary carcinogenesis as driven by subcutaneous slow-release medroxyprogesterone acetate (MPA, M) pellets plus orally administered 7,12-dimethylbenz[ a ]anthracene (DMBA, D), an in vivo model that recapitulates multiple facets of the biology and immunology of human hormone receptor positive (HR + ) breast cancer. Our data demonstrate that female mice bearing a whole-body, homozygous deletion in peptidylprolyl isomerase F ( Ppif ), which encodes a key regulator of MPT-driven necrosis commonly known as CYPD, but not female mice with systemic defects in necroptosis as imposed by the whole body-deletion homozygous of receptor-interacting serine-threonine kinase 3 ( Ripk3 ) or mixed lineage kinase domain like pseudokinase ( Mlkl ), are more susceptible to M/D-driven carcinogenesis than their wild-type counterparts. These findings point to CYPD as to an oncosuppressive protein that restrains HR + mammary carcinogenesis in mice, at least potentially via MPT-driven necrosis. Biological sciences/Cancer/Breast cancer Biological sciences/Cell biology/Cell death cyclosporin A immunogenic cell death immunosurveillance inflammasome permeability transition pore complex Figures Figure 1 Figure 2 Figure 3 Introduction Mammalian cells are equipped with a variety of mechanisms that ensure their controlled demise in both physiological and pathological settings 1 , 2 , 3 . Indeed, while for a long time apoptosis was believed to be the sole cell death pathway to be genetically controlled, it is now widely accepted that mammalian cells can also undergo various regulated forms of necrosis 1 , 2 , 3 . These include (but are not limited to): (1) mitochondrial permeability transition (MPT)-driven necrosis, which is precipitated by peptidylprolyl isomerase F (PPIF, also known as CYPD) 4 , and (2) necroptosis, which requires the kinase activity of receptor-interacting serine-threonine kinase 3 (RIPK3) as well as the ability of mixed lineage kinase domain like pseudokinase (MLKL) to form pores in the plasma membrane 5 , 6 . Importantly, both these cell death subroutines can drive inflammation if not be overtly immunogenic (i.e., elicit antigen-specific immune responses associated with immunological memory) 7 , 8 , 9 , at least in part reflecting: (1) the ability of multiple mitochondrial components to drive inflammation once released in the cytosol downstream of MPT 10 , and (2) the ability of RIPK3 to engage inflammasome signaling and hence promote the maturation and release of interleukin 1 beta (IL1B) and IL18 (Ref. 6 ). Thus, at least a priori , pre-malignant cells undergoing MPT-driven necrosis or necroptosis as a consequence of adverse microenvironmental conditions may elicit inflammatory processes or adaptive immune responses that drive 11 , 12 or restrain 8 tumor progression, respectively. We harnessed female C57BL/6J mice bearing whole-body, homozygous deletions in Ppif , Ripk3 or Mlkl to test whether systemic defects in MPT-driven necrosis or necroptosis influence mammary carcinogenesis as elicited by the subcutaneous implantation of slow-release medroxyprogesterone acetate (MPA, M) pellets coupled with the oral administration of 7,12-dimethylbenz[ a ]anthracene (DMBA, D) 13 , 14 , 15 . We deliberately chose this mouse model of HR + mammary carcinogenesis because of its unique immunobiological resemblance to its human counterpart. Besides sharing transcriptional features with human HR + HER2 − breast cancer 13 , M/D-driven mammary carcinomas established in immunocompetent female C57BL/6J mice are indeed poorly infiltrated by immune cells at baseline, and hence are poorly responsive to immune checkpoint inhibitors specific for PD-1 13 , but exquisitely sensitive to CDK4/6 inhibitors 14 , similar to their human counterparts 16 , 17 , 18 . Moreover, M/D-driven mammary carcinogenesis appears to be susceptible to risk factors similarly increasing the propensity of postmenopausal women to develop HR + breast cancer, such as obesity 13 , 19 . Finally, M/D-driven mammary carcinomas not only fail to express erb-b2 receptor tyrosine kinase 2 (ERBB2, best known as HER2), but most often also preserve estrogen receptor 1 (ESR1) and progesterone receptor (PGR) expression throughout the oncogenic process 13 , hence exhibiting fundamental differences from other mouse models of breast cancer expressing HRs such as MMTV-PyMT mice. Indeed, the latter robustly express HER2 and tend to lose HR expression by the time mice are randomized to treatment or tumors are collected to generate cell lines 20 , 21 , 22 , de facto modeling another type (HER2 + ) of breast cancer. We found that female Ppif −/− mice, but not their Ripk3 −/− or Mlkl −/− counterparts, develop M/D-driven mammary carcinomas with a shorter delay than wild-type (WT) mice at both primary and secondary disease sites, resulting in reduced overall survival despite a comparable growth of established tumors. These findings indicate that CYPD restrains the initial steps of HR + mammary carcinogenesis in mice, at least potentially through its fundamental role in the control of MPT-driven necrosis. Materials and Methods Mice and oncogenesis. Animal studies were performed as per guidelines from the Guide for the Care and Use of Laboratory Animals 23 and under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Weill Cornell Medical College (n° 2020-0022). Endogenous mammary carcinogenesis was initiated as previously described 13 , 24 . Shortly, a 50 mg slow-release (90 days) medroxyprogesterone acetate (MPA, M) pellet (#NP-161, Innovative Research of America) was implanted subcutaneously in the interscapular area of 6–9 weeks old female C57BL/6J mice (Jackson). One week later, mice received 1 mg 7,12-dimethylbenz[a]anthracene (DMBA, D) in 200 µL corn oil (#C8267, Millipore Sigma) by oral gavage, a procedure that was repeated on weeks 2, 3, 5, 6 and 7 after implantation of the MPA pellet 13 , 24 . Mice were routinely checked for the appearance of mammary lesions, which were monitored for growth with a common caliper. Mice were euthanatized when the cumulative surface of all neoplastic lesions (computed as the area of an ellipse: A = longest diameter X shortest diameter X π/4) reached 180–200 mm 2 (ethical endpoint that was employed as surrogate marker for survival), or in the context of evident toxicity or distress ( e.g. , hunching, anorexia, tumor ulceration). Data processing and statistical analysis. The following parameters were measured or scored: (1) tumor-free survival (TFS), defined as the number of days between the 1st DMBA administration and the detection of the first malignant lesion; (2) time to secondary disease (TT2), defined as the number of days between the detection of the first malignant lesions and the detection of any other malignant lesion; (3) time to death (TTD), defined as the number of days between the detection of the first malignant lesions and ethical endpoint (see above); (4) overall survival (OS), defined as the number of days between the 1st DMBA administration and ethical endpoint (see above); (5) number of secondary tumors at euthanasia; (6) normalized number of secondary tumors at euthanasia, defined as the number of secondary tumors at euthanasia divided by TTD; (7) % of primary tumor burden (T1) at euthanasia, defined as follows: T1 (%) = 100 X surface area of the primary tumor / surface area of all tumors; (8) primary tumor growth; (9) secondary tumor growth; (10) cumulative tumor growth. Prism v. 10.2.3 (GraphPad) and Excel 2021 (Microsoft) were used for data processing, plotting and statistical analysis. Illustrator 2025 (Adobe) was used for figure preparation. One-way ANOVA plus Geisser-Greenhouse correction and Fisher’s LSD were applied to assess statistical significance in comparisons involving numerical data. Incidence of secondary oncogenesis was assessed for statistical significance by Fisher’s exact test. Growth curves were assessed for statistical significance by two-way ANOVA plus Geisser-Greenhouse correction. TFS, TT2, TTD and OS curves were assessed for statistical significance by Log-rank (Mantel-Cox) and Mantel-Haenszel tests. Whenever relevant, number of mice per group, hazard ratio (HR) plus 95% confidence interval (CI) and p values are reported. Results Ppif restrains primary M/D-driven mammary carcinogenesis. To elucidate the impact of systemic defects in MPT-driven necrosis and necroptosis on HR + mammary carcinogenesis, we subjected female WT, Ppif −/− , Ripk3 −/− and Mlkl −/− C57BL/6J mice of 6–9 weeks of age to M/D-driven mammary carcinogenesis according to established procedures 13 , 24 , and monitored them for tumor-free survival (TFS), as well as for a number of other parameters defining disease progression (Fig. 1 A). In line with previous findings from us and others 13 , 25 , female WT mice developed M/D-driven mammary carcinomas with complete penetrance and a median latency of 89 days from the 1st DMBA gavage (Fig. 1 B). Neither the Ripk3 −/− nor the Mlkl −/− genotype significantly influenced tumor penetrance (data not shown) or latency in this setting (median TFS: 104 days and 83 days, respectively; p value: 0.9064 and 0.1875, respectively) (Fig. 1 B). Conversely, while Ppif −/− mice also developed M/D-driven mammary tumors with complete penetrance, they exhibited a significantly reduced TFS as compared to WT mice (median TFS: 69 days; p value: 0.0213) (Fig. 1 B). However, the growth of first detectable (primary) M/D-driven carcinomas, as monitored from tumor detection with a common caliper, did not differ between Ppif −/− and WT mice ( p value: 0. 678), while it was slightly (but significantly) reduced in their Ripk3 −/− and Mlkl −/− genotype ( p value: 0.022 and < 0.0001, respectively) (Fig. 1 C). These findings demonstrate that the whole-body homozygous deletion of Ppif shortens the latency for M/D-driven mammary carcinomas to become detectable in the absence of overt alterations in tumor growth rate. Lack of Ppif promotes secondary M/D-driven mammary carcinogenesis. M/D-driven oncogenesis proceeds beyond the formation of detectable primary tumors, resulting in the appearance of extra (secondary) lesions that contribute to cumulative tumor burden and hence to the definition of humane endpoint 15 . To understand the impact of genetic alterations in key molecular regulators of MPT-driven necrosis and necroptosis, we thus assessed time to secondary oncogenesis (TT2), defined as the number of days elapsing between the detection of the primary M/D-driven tumor and any extra mammary lesions emerging thereafter. Most often, WT mice had to be euthanatized because of the uncontrolled growth of primary M/D-driven tumors before developing a secondary neoplasm, hence failing to reach median TT2 (Fig. 2 A). Indeed, only 6/22 (~ 27.3%) mice in this group developed at least one secondary tumor by the time euthanasia was required owing to global disease burden (Fig. 2 B). Ripk3 −/− and Mlkl −/− mice exhibited a median TT2 of 23 and 39 days, respectively, which was not significantly different compared to WT mice ( p value: 0.3847 and 0.9165, respectively) (Fig. 2 A). Accordingly, 4/8 (50%) Ripk3 −/− mice and 4/7 (~ 57.1%) Mlkl −/− mice developed at least one secondary lesion before global tumor burden reached ethical endpoint, which failed to differ in a statistically significant manner from WT mice ( p value: 0.3841 and 0.1476, respectively) (Fig. 2 B). Conversely, secondary M/D-driven tumorigenesis exhibited a strong (although sub-significant) trend towards acceleration in Ppif −/− mice (median TT2: 7 days; p value: 0.0763), and these animals developed at least one secondary tumor in 7/11 cases (~ 63.6%, p value: 0.0436) (Fig. 2 A,B). Of note, secondary M/D-driven tumors failed to exhibit differences in growth pattern when Ripk3 −/− mice were compared to their WT counterparts in this respect ( p value: 0.4020) (Fig. 2 C). Conversely, while secondary M/D-driven carcinomas evolving in Mlkl −/− mice grew less rapidly compared to the same tumors progressing in WT mice ( p value: <0.0001), the contrary was true for secondary M/D-driven tumors developing in Ppif −/− mice ( p value: 0.0003) (Fig. 2 C). Finally, Ripk3 −/− mice and Mlkl −/− mice did not differ from WT mice with respect to the number of secondary M/D-driven tumors per mouse ( p value: 0.3230 and 0.1169, respectively), even when this parameter was normalized for mouse survival ( p value: 0.5101 and 0.3945, respectively) (Fig. 2 D,E). On the contrary, Ppif −/− mice subjected to M/D-driven carcinogenesis accumulated – in average – an increased amount of secondary lesions per mouse as compared to WT mice, not only as an absolute measurement ( p value: 0.0175), but also upon accounting for differential survival ( p value: 0.0176) (Fig. 2 D,E). Collectively, these data demonstrate that the whole-body deletion of Ppif accelerates HR + carcinogenesis as driven in C57BL/6J mice by MPA and DMBA not only at primary, but also at secondary, disease sites. Ppif inhibits natural disease progression in M/D-driven mammary carcinomas. Despite the early appearance of primary and secondary M/D-driven tumors as well as the accelerated tumor growth at secondary disease sites as documented in female Ppif −/− mice (Fig. 1 B and 2 A-C), these animals exhibited a median time to death (TDD), defined as the number of days elapsing between the detection of the first malignant lesion and ethical endpoint as dictated by cumulative tumor burden, of 17 days, which was not significantly different from that of WT mice (median TTD: 11 days, p value: 0.2593) (Fig. 3 A), potentially owing to a slight (although sub-significant) deceleration in primary tumor growth (Fig. 1 C). In line with this notion, the Ripk3 −/− and even more so the Mlkl −/− genotype were associated with an extension in TTD (median TTD: 25 and 30 days, respectively; p value: 0.0084 and 0.0035, respectively) (Fig. 3 A), largely reflecting the reduced speed of tumor progression at primary disease sites (Fig. 1 B) in the context of limited alterations in TT2 and secondary tumor growth (Fig. 2 A-C). Consistent with this notion, while the growth of all detectable M/D-driven tumors failed to differ between WT and Ppif −/− mice ( p value: 0.1914), both the Ripk3 −/− and the Mlkl −/− genotype ( p value: 0.0086 and < 0.0001, respectively) were associated with significant reduction in global disease progression (Fig. 3 B). Of note, the relative contribution of primary disease to global tumor burden as a determinant of ethical endpoint was not affected by the whole-body deletion of Ripk3 ( p value: 0.1780) or Mlkl ( p value: 0.2056) (Fig. 3 C). Conversely, the Ppif −/− genotype tended to be associated (although in a sub-significant manner) with a decreased relative contribution of primary over secondary tumors to global disease burden at ethical endpoint ( p value: 0.0809) (Fig. 3 C). Moreover, while both the Ripk3 −/− and the Mlkl −/− genotype failed to influence the overall survival (OS) of female mice subjected to M/D-driven carcinogenesis (median OS: 125 and 119 days, respectively; p value: 0.2679 and 0.7347, respectively), the whole-body deletion of Ppif significantly shortened it (median OS: 90 days; p value: 0.0330), with WT animals exhibiting a median OS of 100 days (Fig. 3 D). Taken together, these data indicate that CYPD restrains the natural progression of HR + mammary carcinogenesis in female C57BL/6J mice by interfering with early stages of tumorigenesis Discussion In summary, our data indicate that CYPD – a fundamental regulator of MPT-driven necrosis 26 , 27 – mediates oncosuppressive effects in an immunocompetent mouse model of HR + mammary oncogenesis driven by the systemic administration of a chemical carcinogen, i.e. , DMBA, in the context of supraphysiological PGR signaling, as elicited by slow-release MPA pellets. 13 , 14 , 15 As introduced above, this is a uniquely translational model of HR + HER2 − oncogenesis, as it recapitulates a number of biological, immunological and therapeutic aspects of its human counterpart, 13 , 14 , 15 hence standing out as a preferential platform for immuno-oncology studies of this specific variant of breast cancer. 28 , 29 Moreover, our findings are fully in line with the well-recognized oncosuppressive role of regulated cell death (RCD) in many of its variants, 30 , 31 , 32 largely (but perhaps not exclusively) reflecting the evolutionary advantage provided to a multicellular organism by signal transduction cascades that coordinate the demise of individual cells bearing excessive macromolecular damage (hence being unable to perform their physiological functions or even at increased risk of malignant transformation) in the context of adequate immunological responses. 33 , 34 CYPD has been shown to mediate various functions that may or may not involve MPT regulation but definitely do not culminate with MPT-driven necrosis, including a paradoxical cytoprotective function in senescent cells. 35 , 36 Thus, it remains possible that the ability of CYPD to suppress HR + mammary carcinogenesis in female C57BL/6J mice may be unrelated to RCD via MPT-driven necrosis. This is particularly challenging to formally establish with additional genetic approaches, 37 as most (if not all) proteins that reportedly form or interact with – hence regulating – the supramolecular complex responsible for MPT, which is commonly known the permeability transition pore complex (PTPC): (1) exhibit considerable genetic and/or functional redundancy, considerably complicating the implementation of successful knockout strategies in vivo ; 38 , 39 (2) are critical components of the molecular machinery that ensure mitochondrial ATP synthesis, de facto being strictly required for survival; 40 , 41 , 42 and (3) at least in some cases, have been conclusively shown to be dispensable for MPT. 26 , 43 , 44 Along similar lines, currently available pharmacological inhibitors of the MPT exhibit limited specificity. 37 As a standalone example, the pharmacological CYPD inhibitor cyclosporin A (CsA) has major CYPD-independent immunosuppressive effects by inhibiting peptidylprolyl isomerase A (PPIA, best known as CYPA) in T cells. 45 , 46 Intriguingly, CYPD has also been shown to contribute to normal T cell and natural killer (NK) cell functions, at least in preclinical models of infection, 47 , 48 raising the possibility that accelerated MPA/DMBA-driven mammary carcinogenesis as observed in Ppif −/− C57BL/6J mice may result from defects in natural immunosurveillance. 49 We have previously demonstrated that MPA/DMBA-driven mammary tumors develop with an accelerated kinetic in Rag2 −/− Il2rg −/− mice (which lack T cells, B cells and NK cells), as well as in mice receiving an antibody specific for NKG2D (which depletes NK cells and a subpopulation of CD8 + T cells), but not in Rag2 −/− mice (which lack T and B cells) or in mice receiving CD4- and CD8-targeting antibodies (which are depleted of T cells), globally pointing to NK cells as to central mediators of natural immunosurveillance in this model. 13 , 50 Subjecting C57BL/6J mice to total body irradiation (TBI)-induced myeloablation and reconstituting them with Ppif −/− hematopoietic stem cells (and vice versa) will provide additional insights into the role of CYPD expression in radiosensitive vs radioresistant cells in MPA/DMBA-driven mammary carcinogenesis. Despite this and other open avenues, our findings indicate that CYPD retards HR + mammary carcinogenesis in immunocompetent C57BL/6J mice. Of note, CYPD has previously been shown to promote (rather than inhibit) hepatocellular carcinogenesis in mice with non-alcoholic steatohepatitis (NASH) 51 as well HR + HER2 + mammary carcinogenesis as driven by the MMTV-PyMT construct. 52 Additional work is hence required to understand whether our data reflect unique immunobiological features of HR + breast cancer over other breast cancer subtypes and extramammary neoplasms. Declarations Author contributions. AB performed experimental assessments with help from AS, GP and CG, under supervision from LG. AB and MBV analyzed the data and prepared figures under supervision from LG. LG wrote the manuscript with constructive input from all authors. All authors approve the submitted version of the article. Competing Interests. LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. All other authors have no conflicts to declare. Acknowledgements. Ppif -/- , Ripk3 -/- and Mlkl -/- mice were a generous gift of Dr. Augustine M.K. Choi (Weill Cornell Medical College, New York, US). The AB lab is/has been supported (as a PI unless otherwise indicated) a Collaborative Research Initiative Grant from the Sandra and Edward Meyer Cancer Center (New York, US) and by startup funds from Fox Chase Cancer Center (Philadelphia, US). The LG lab is/has been supported (as a PI unless otherwise indicated) by one NIH R01 grant (#CA271915), by two Breakthrough Level 2 grants from the US DoD BCRP (#BC180476P1, #BC210945), by a grant from the STARR Cancer Consortium (#I16-0064), by a Transformative Breast Cancer Consortium Grant from the US DoD BCRP (#W81XWH2120034, PI: Formenti), by a U54 grant from NIH/NCI (#CA274291, PI: Deasy, Formenti, Weichselbaum), by the 2019 Laura Ziskin Prize in Translational Research (#ZP-6177, PI: Formenti) from the Stand Up to Cancer (SU2C), by a Mantle Cell Lymphoma Research Initiative (MCL-RI, PI: Chen-Kiang) grant from the Leukemia and Lymphoma Society (LLS), by a Rapid Response Grant from the Functional Genomics Initiative (New York, US), by a pre-SPORE grant (PI: Demaria, Formenti), a Collaborative Research Initiative Grant and a Clinical Trials Innovation Grant from the Sandra and Edward Meyer Cancer Center (New York, US), by startup funds from the Dept. of Radiation Oncology at Weill Cornell Medicine (New York, US), by startup funds from Fox Chase Cancer Center (Philadelphia, US), by industrial collaborations with Lytix Biopharma (Oslo, Norway), Promontory (New York, US) and Onxeo (Paris, France), as well as by donations from Promontory (New York, US), the Luke Heller TECPR2 Foundation (Boston, US), Sotio a.s. 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Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol 2007, 9(5): 550–555. Galluzzi L, Kroemer G. Mitochondrial apoptosis without VDAC. Nat Cell Biol 2007, 9(5): 487–489. Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 1984, 226(4674): 544–547. Colombani PM, Robb A, Hess AD. Cyclosporin A binding to calmodulin: a possible site of action on T lymphocytes. Science 1985, 228(4697): 337–339. Tzelepis F, Blagih J, Khan N, Gillard J, Mendonca L, Roy DG, et al. Mitochondrial cyclophilin D regulates T cell metabolic responses and disease tolerance to tuberculosis. Sci Immunol 2018, 3(23). Downey J, Randolph HE, Pernet E, Tran KA, Khader SA, King IL, et al. Mitochondrial cyclophilin D promotes disease tolerance by licensing NK cell development and IL-22 production against influenza virus. Cell Rep 2022, 39(12): 110974. Kroemer G, Chan TA, Eggermont AMM, Galluzzi L. Immunosurveillance in clinical cancer management. CA Cancer J Clin 2024, 74(2): 187–202. Buque A, Bloy N, Petroni G, Kroemer G, Galluzzi L. NK cells beat T cells at early breast cancer control. Oncoimmunology 2020, 9(1): 1806010. Stauffer WT, Bobardt M, Ure DR, Foster RT, Gallay P. Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH. PLoS One 2024, 19(4): e0301711. Bigi A, Beltrami E, Trinei M, Stendardo M, Pelicci PG, Giorgio M. Cyclophilin D counteracts P53-mediated growth arrest and promotes Ras tumorigenesis. Oncogene 2016, 35(39): 5132–5143. Additional Declarations There is a conflict of interest LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. All other authors have no conflicts to declare. Cite Share Download PDF Status: Published Journal Publication published 10 Jun, 2025 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 31 Mar, 2025 Review # 1 received at journal 28 Mar, 2025 Reviewer # 1 agreed at journal 20 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Submission checks completed at journal 20 Mar, 2025 Editor assigned by journal 19 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6265353","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":431677245,"identity":"f235f6c3-55c4-42b6-8a70-35b518bec049","order_by":0,"name":"Lorenzo Galluzzi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYDCCAwwMEgwMNjz8IE5CAfFa0uQkG0BaDIjXctjY4ACIR4wWvuPND298qElL3Hx+deKHBwYM8vxiB/BrkTxzzNhyxjGbxG033m6WADrMcObsBPxaDG4kmEnzNqQBtZzdANKSYHCbkJb7z78BtRxO3Dzj7OYfxGm5wQOyBeh9/t5txNkieSanGOiXNDmJG7zbLBIMJAj7he/48Y3AEANGZf/ZzTd/VNjI80sT0IIAEmCVEsQqBwH+A6SoHgWjYBSMgpEEAMc3Se9GjoqOAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2257-8500","institution":"Fox Chase Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Galluzzi","suffix":""},{"id":431677246,"identity":"5f468091-eca8-4397-beb9-406046b6fe19","order_by":1,"name":"Aitziber Buqué","email":"","orcid":"","institution":"Fox Chase Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Aitziber","middleName":"","lastName":"Buqué","suffix":""},{"id":431677247,"identity":"98fafa64-1447-44a3-955b-4dc7a732d8f6","order_by":2,"name":"Manuel Beltrán-Visiedo","email":"","orcid":"","institution":"Fox Chase Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Beltrán-Visiedo","suffix":""},{"id":431677248,"identity":"a93ce50d-5f05-4dc8-924a-cd6dd1a8c002","order_by":3,"name":"Ai Sato","email":"","orcid":"","institution":"Weill Cornell Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ai","middleName":"","lastName":"Sato","suffix":""},{"id":431677249,"identity":"df038003-5b8b-4601-bbaf-61fe093f61b1","order_by":4,"name":"Claudia Galassi","email":"","orcid":"","institution":"Weill Cornell Medical College","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Galassi","suffix":""},{"id":431677250,"identity":"c96af191-cff8-4917-9794-cac9de9765f7","order_by":5,"name":"Giulia Petroni","email":"","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Petroni","suffix":""}],"badges":[],"createdAt":"2025-03-20 02:15:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6265353/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6265353/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-025-02555-0","type":"published","date":"2025-06-10T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79564604,"identity":"b4ce8550-4911-44d5-8a86-0f6cc9d55fcc","added_by":"auto","created_at":"2025-03-31 09:19:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1589278,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e restrains primary M/D-driven mammary carcinogenesis. \u003c/strong\u003eWild-type (WT) \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e female C57BL/6J mice were subjected M/D-driven carcinogenesis, then assessed for tumor-free survival (TFS), time to secondary disease (TT2), time to death (TTD) and overall survival (OS), as well as routinely monitored for tumor growth at primary and secondary disease sites (\u003cstrong\u003eA\u003c/strong\u003e). TFS (\u003cstrong\u003eB\u003c/strong\u003e) and tumor growth at primary disease sites (\u003cstrong\u003eC\u003c/strong\u003e) are reported. In \u003cstrong\u003eB\u003c/strong\u003e, median TFS, Mantel-Haenszel hazard ratio (HR) with 95% confidence interval (CI), group size (n) and \u003cem\u003ep\u003c/em\u003e values (Log-rank, compared to WT mice) are indicated. In \u003cstrong\u003eC\u003c/strong\u003e, both individual and average tumor growth are illustrated, with group size (n) and \u003cem\u003ep\u003c/em\u003e values (2-way ANOVA, compared to WT mice) reported.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6265353/v1/d72b04b1b1a0ca37d79b350b.png"},{"id":79564602,"identity":"85f2feb6-f6fe-46f8-b1b4-5c15447a5760","added_by":"auto","created_at":"2025-03-31 09:19:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1712056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLack of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Ppif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promotes secondary M/D-driven mammary carcinogenesis. \u003c/strong\u003eWild-type (WT) \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e female C57BL/6J mice were subjected M/D-driven carcinogenesis and analyzed as illustrated in \u003cstrong\u003eFig. 1A\u003c/strong\u003e. Time to secondary disease (TT2) (\u003cstrong\u003eA\u003c/strong\u003e), percentage of mice developing secondary lesions (\u003cstrong\u003eB\u003c/strong\u003e), secondary tumor growth (\u003cstrong\u003eC\u003c/strong\u003e), number of secondary tumors per mouse (\u003cstrong\u003eD\u003c/strong\u003e) and number of secondary tumors per mouse normalized to time to death (TTD) (\u003cstrong\u003eE\u003c/strong\u003e) are reported. In \u003cstrong\u003eA\u003c/strong\u003e, median TT2, Mantel-Haenszel hazard ratio (HR) with 95% confidence interval (CI), group size (n) and \u003cem\u003ep\u003c/em\u003e values (Log-rank) are indicated. Mice succumbing to primary disease without developing a secondary tumor were censored from the analysis. In \u003cstrong\u003eB\u003c/strong\u003e, group size (n) and \u003cem\u003ep\u003c/em\u003e values (Fisher’s exact test, compared to WT mice) are indicated. In \u003cstrong\u003eC\u003c/strong\u003e, both individual and average tumor growth are illustrated, with group size (n) and \u003cem\u003ep\u003c/em\u003e values (2-way ANOVA, compared to WT mice) reported. In \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003e, results are reported as means ± SEM and individual data points, \u003cem\u003ep\u003c/em\u003e values (Kruskal-Wallis, compared to WT mice) are indicated. NR, not reached.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6265353/v1/0f7885db2a09a7b1e8456a80.png"},{"id":79564600,"identity":"4915a5b5-de18-474b-a7d6-09986100aae0","added_by":"auto","created_at":"2025-03-31 09:19:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2246660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePpif\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inhibits natural disease progression in M/D-driven mammary carcinomas. \u003c/strong\u003eWild-type (WT) \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e female C57BL/6J mice were subjected M/D-driven carcinogenesis and analyzed as illustrated in \u003cstrong\u003eFig. 1A\u003c/strong\u003e. Time to death (TTD) (\u003cstrong\u003eA\u003c/strong\u003e), cumulative tumor growth (\u003cstrong\u003eB\u003c/strong\u003e), relative contribution of primary tumors (T1) to overall disease burden at euthanasia (\u003cstrong\u003eC\u003c/strong\u003e) and overall survival (OS) (\u003cstrong\u003eD\u003c/strong\u003e) are illustrated. In \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e, median values, Mantel-Haenszel hazard ratio (HR) with 95% confidence interval (CI), group size (n) and \u003cem\u003ep\u003c/em\u003e values (Log-rank) are indicated. Mice succumbing to causes other than euthanasia owing to global disease burden were censored from the analysis. In \u003cstrong\u003eB\u003c/strong\u003e, both individual and average tumor growth are illustrated, with group size (n) and \u003cem\u003ep\u003c/em\u003e values (2-way ANOVA, compared to WT mice) reported. In \u003cstrong\u003eC\u003c/strong\u003e, results are reported as means ± SEM and individual data points, \u003cem\u003ep\u003c/em\u003e values (Kruskal-Wallis, compared to WT mice) are indicated.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6265353/v1/b2e3f7a0da8708b46337f8e0.png"},{"id":84370757,"identity":"173769d1-87ab-45de-a5b1-e0e1c83b16e8","added_by":"auto","created_at":"2025-06-11 07:09:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6330733,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6265353/v1/354fed68-74bb-443c-8ee3-2cae5485a4c3.pdf"}],"financialInterests":"There is a conflict of interest\nLG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. All other authors have no conflicts to declare.","formattedTitle":"\u003cp\u003eCYPD limits HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis in mice\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMammalian cells are equipped with a variety of mechanisms that ensure their controlled demise in both physiological and pathological settings\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Indeed, while for a long time apoptosis was believed to be the sole cell death pathway to be genetically controlled, it is now widely accepted that mammalian cells can also undergo various regulated forms of necrosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These include (but are not limited to): (1) mitochondrial permeability transition (MPT)-driven necrosis, which is precipitated by peptidylprolyl isomerase F (PPIF, also known as CYPD)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and (2) necroptosis, which requires the kinase activity of receptor-interacting serine-threonine kinase 3 (RIPK3) as well as the ability of mixed lineage kinase domain like pseudokinase (MLKL) to form pores in the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Importantly, both these cell death subroutines can drive inflammation if not be overtly immunogenic (i.e., elicit antigen-specific immune responses associated with immunological memory)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, at least in part reflecting: (1) the ability of multiple mitochondrial components to drive inflammation once released in the cytosol downstream of MPT\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and (2) the ability of RIPK3 to engage inflammasome signaling and hence promote the maturation and release of interleukin 1 beta (IL1B) and IL18 (Ref. \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e). Thus, at least \u003cem\u003ea priori\u003c/em\u003e, pre-malignant cells undergoing MPT-driven necrosis or necroptosis as a consequence of adverse microenvironmental conditions may elicit inflammatory processes or adaptive immune responses that drive\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e or restrain\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e tumor progression, respectively.\u003c/p\u003e \u003cp\u003eWe harnessed female C57BL/6J mice bearing whole-body, homozygous deletions in \u003cem\u003ePpif\u003c/em\u003e, \u003cem\u003eRipk3\u003c/em\u003e or \u003cem\u003eMlkl\u003c/em\u003e to test whether systemic defects in MPT-driven necrosis or necroptosis influence mammary carcinogenesis as elicited by the subcutaneous implantation of slow-release medroxyprogesterone acetate (MPA, M) pellets coupled with the oral administration of 7,12-dimethylbenz[\u003cem\u003ea\u003c/em\u003e]anthracene (DMBA, D)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We deliberately chose this mouse model of HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis because of its unique immunobiological resemblance to its human counterpart. Besides sharing transcriptional features with human HR\u003csup\u003e+\u003c/sup\u003eHER2\u003csup\u003e\u0026minus;\u003c/sup\u003e breast cancer\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, M/D-driven mammary carcinomas established in immunocompetent female C57BL/6J mice are indeed poorly infiltrated by immune cells at baseline, and hence are poorly responsive to immune checkpoint inhibitors specific for PD-1\u003csup\u003e13\u003c/sup\u003e, but exquisitely sensitive to CDK4/6 inhibitors\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, similar to their human counterparts\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Moreover, M/D-driven mammary carcinogenesis appears to be susceptible to risk factors similarly increasing the propensity of postmenopausal women to develop HR\u003csup\u003e+\u003c/sup\u003e breast cancer, such as obesity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Finally, M/D-driven mammary carcinomas not only fail to express erb-b2 receptor tyrosine kinase 2 (ERBB2, best known as HER2), but most often also preserve estrogen receptor 1 (ESR1) and progesterone receptor (PGR) expression throughout the oncogenic process\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, hence exhibiting fundamental differences from other mouse models of breast cancer expressing HRs such as MMTV-PyMT mice. Indeed, the latter robustly express HER2 and tend to lose HR expression by the time mice are randomized to treatment or tumors are collected to generate cell lines\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ede facto\u003c/em\u003e modeling another type (HER2\u003csup\u003e+\u003c/sup\u003e) of breast cancer.\u003c/p\u003e \u003cp\u003eWe found that female \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, but not their \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e counterparts, develop M/D-driven mammary carcinomas with a shorter delay than wild-type (WT) mice at both primary and secondary disease sites, resulting in reduced overall survival despite a comparable growth of established tumors. These findings indicate that CYPD restrains the initial steps of HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis in mice, at least potentially through its fundamental role in the control of MPT-driven necrosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eMice and oncogenesis.\u003c/b\u003e Animal studies were performed as per guidelines from the Guide for the Care and Use of Laboratory Animals\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Weill Cornell Medical College (n\u0026deg; 2020-0022). Endogenous mammary carcinogenesis was initiated as previously described\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Shortly, a 50 mg slow-release (90 days) medroxyprogesterone acetate (MPA, M) pellet (#NP-161, Innovative Research of America) was implanted subcutaneously in the interscapular area of 6\u0026ndash;9 weeks old female C57BL/6J mice (Jackson). One week later, mice received 1 mg 7,12-dimethylbenz[a]anthracene (DMBA, D) in 200 \u0026micro;L corn oil (#C8267, Millipore Sigma) by oral gavage, a procedure that was repeated on weeks 2, 3, 5, 6 and 7 after implantation of the MPA pellet\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Mice were routinely checked for the appearance of mammary lesions, which were monitored for growth with a common caliper. Mice were euthanatized when the cumulative surface of all neoplastic lesions (computed as the area of an ellipse: A\u0026thinsp;=\u0026thinsp;longest diameter X shortest diameter X π/4) reached 180\u0026ndash;200 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e (ethical endpoint that was employed as surrogate marker for survival), or in the context of evident toxicity or distress (\u003cem\u003ee.g.\u003c/em\u003e, hunching, anorexia, tumor ulceration).\u003c/p\u003e \u003cp\u003e \u003cb\u003eData processing and statistical analysis.\u003c/b\u003e The following parameters were measured or scored: (1) tumor-free survival (TFS), defined as the number of days between the 1st DMBA administration and the detection of the first malignant lesion; (2) time to secondary disease (TT2), defined as the number of days between the detection of the first malignant lesions and the detection of any other malignant lesion; (3) time to death (TTD), defined as the number of days between the detection of the first malignant lesions and ethical endpoint (see above); (4) overall survival (OS), defined as the number of days between the 1st DMBA administration and ethical endpoint (see above); (5) number of secondary tumors at euthanasia; (6) normalized number of secondary tumors at euthanasia, defined as the number of secondary tumors at euthanasia divided by TTD; (7) % of primary tumor burden (T1) at euthanasia, defined as follows: T1 (%)\u0026thinsp;=\u0026thinsp;100 X surface area of the primary tumor / surface area of all tumors; (8) primary tumor growth; (9) secondary tumor growth; (10) cumulative tumor growth. Prism v. 10.2.3 (GraphPad) and Excel 2021 (Microsoft) were used for data processing, plotting and statistical analysis. Illustrator 2025 (Adobe) was used for figure preparation. One-way ANOVA plus Geisser-Greenhouse correction and Fisher\u0026rsquo;s LSD were applied to assess statistical significance in comparisons involving numerical data. Incidence of secondary oncogenesis was assessed for statistical significance by Fisher\u0026rsquo;s exact test. Growth curves were assessed for statistical significance by two-way ANOVA plus Geisser-Greenhouse correction. TFS, TT2, TTD and OS curves were assessed for statistical significance by Log-rank (Mantel-Cox) and Mantel-Haenszel tests. Whenever relevant, number of mice per group, hazard ratio (HR) plus 95% confidence interval (CI) and \u003cem\u003ep\u003c/em\u003e values are reported.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003erestrains primary M/D-driven mammary carcinogenesis.\u003c/b\u003e To elucidate the impact of systemic defects in MPT-driven necrosis and necroptosis on HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis, we subjected female WT, \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e C57BL/6J mice of 6\u0026ndash;9 weeks of age to M/D-driven mammary carcinogenesis according to established procedures\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and monitored them for tumor-free survival (TFS), as well as for a number of other parameters defining disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In line with previous findings from us and others\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, female WT mice developed M/D-driven mammary carcinomas with complete penetrance and a median latency of 89 days from the 1st DMBA gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Neither the \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e nor the \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotype significantly influenced tumor penetrance (data not shown) or latency in this setting (median TFS: 104 days and 83 days, respectively; \u003cem\u003ep\u003c/em\u003e value: 0.9064 and 0.1875, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Conversely, while \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice also developed M/D-driven mammary tumors with complete penetrance, they exhibited a significantly reduced TFS as compared to WT mice (median TFS: 69 days; \u003cem\u003ep\u003c/em\u003e value: 0.0213) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, the growth of first detectable (primary) M/D-driven carcinomas, as monitored from tumor detection with a common caliper, did not differ between \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice (\u003cem\u003ep\u003c/em\u003e value: 0. 678), while it was slightly (but significantly) reduced in their \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotype (\u003cem\u003ep\u003c/em\u003e value: 0.022 and \u0026lt;\u0026thinsp;0.0001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings demonstrate that the whole-body homozygous deletion of \u003cem\u003ePpif\u003c/em\u003e shortens the latency for M/D-driven mammary carcinomas to become detectable in the absence of overt alterations in tumor growth rate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLack of\u003c/b\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003epromotes secondary M/D-driven mammary carcinogenesis.\u003c/b\u003e M/D-driven oncogenesis proceeds beyond the formation of detectable primary tumors, resulting in the appearance of extra (secondary) lesions that contribute to cumulative tumor burden and hence to the definition of humane endpoint\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To understand the impact of genetic alterations in key molecular regulators of MPT-driven necrosis and necroptosis, we thus assessed time to secondary oncogenesis (TT2), defined as the number of days elapsing between the detection of the primary M/D-driven tumor and any extra mammary lesions emerging thereafter. Most often, WT mice had to be euthanatized because of the uncontrolled growth of primary M/D-driven tumors before developing a secondary neoplasm, hence failing to reach median TT2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Indeed, only 6/22 (~\u0026thinsp;27.3%) mice in this group developed at least one secondary tumor by the time euthanasia was required owing to global disease burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited a median TT2 of 23 and 39 days, respectively, which was not significantly different compared to WT mice (\u003cem\u003ep\u003c/em\u003e value: 0.3847 and 0.9165, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Accordingly, 4/8 (50%) \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 4/7 (~\u0026thinsp;57.1%) \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice developed at least one secondary lesion before global tumor burden reached ethical endpoint, which failed to differ in a statistically significant manner from WT mice (\u003cem\u003ep\u003c/em\u003e value: 0.3841 and 0.1476, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Conversely, secondary M/D-driven tumorigenesis exhibited a strong (although sub-significant) trend towards acceleration in \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (median TT2: 7 days; \u003cem\u003ep\u003c/em\u003e value: 0.0763), and these animals developed at least one secondary tumor in 7/11 cases (~\u0026thinsp;63.6%, \u003cem\u003ep\u003c/em\u003e value: 0.0436) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf note, secondary M/D-driven tumors failed to exhibit differences in growth pattern when \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were compared to their WT counterparts in this respect (\u003cem\u003ep\u003c/em\u003e value: 0.4020) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Conversely, while secondary M/D-driven carcinomas evolving in \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice grew less rapidly compared to the same tumors progressing in WT mice (\u003cem\u003ep\u003c/em\u003e value: \u0026lt;0.0001), the contrary was true for secondary M/D-driven tumors developing in \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003ep\u003c/em\u003e value: 0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Finally, \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not differ from WT mice with respect to the number of secondary M/D-driven tumors per mouse (\u003cem\u003ep\u003c/em\u003e value: 0.3230 and 0.1169, respectively), even when this parameter was normalized for mouse survival (\u003cem\u003ep\u003c/em\u003e value: 0.5101 and 0.3945, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,E). On the contrary, \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice subjected to M/D-driven carcinogenesis accumulated \u0026ndash; in average \u0026ndash; an increased amount of secondary lesions per mouse as compared to WT mice, not only as an absolute measurement (\u003cem\u003ep\u003c/em\u003e value: 0.0175), but also upon accounting for differential survival (\u003cem\u003ep\u003c/em\u003e value: 0.0176) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,E).\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that the whole-body deletion of \u003cem\u003ePpif\u003c/em\u003e accelerates HR\u003csup\u003e+\u003c/sup\u003e carcinogenesis as driven in C57BL/6J mice by MPA and DMBA not only at primary, but also at secondary, disease sites.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePpif\u003c/b\u003e \u003cb\u003einhibits natural disease progression in M/D-driven mammary carcinomas.\u003c/b\u003e Despite the early appearance of primary and secondary M/D-driven tumors as well as the accelerated tumor growth at secondary disease sites as documented in female \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C), these animals exhibited a median time to death (TDD), defined as the number of days elapsing between the detection of the first malignant lesion and ethical endpoint as dictated by cumulative tumor burden, of 17 days, which was not significantly different from that of WT mice (median TTD: 11 days, \u003cem\u003ep\u003c/em\u003e value: 0.2593) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), potentially owing to a slight (although sub-significant) deceleration in primary tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In line with this notion, the \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and even more so the \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotype were associated with an extension in TTD (median TTD: 25 and 30 days, respectively; \u003cem\u003ep\u003c/em\u003e value: 0.0084 and 0.0035, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), largely reflecting the reduced speed of tumor progression at primary disease sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) in the context of limited alterations in TT2 and secondary tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Consistent with this notion, while the growth of all detectable M/D-driven tumors failed to differ between WT and \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003ep\u003c/em\u003e value: 0.1914), both the \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and the \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotype (\u003cem\u003ep\u003c/em\u003e value: 0.0086 and \u0026lt;\u0026thinsp;0.0001, respectively) were associated with significant reduction in global disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf note, the relative contribution of primary disease to global tumor burden as a determinant of ethical endpoint was not affected by the whole-body deletion of \u003cem\u003eRipk3\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e value: 0.1780) or \u003cem\u003eMlkl\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e value: 0.2056) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Conversely, the \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotype tended to be associated (although in a sub-significant manner) with a decreased relative contribution of primary over secondary tumors to global disease burden at ethical endpoint (\u003cem\u003ep\u003c/em\u003e value: 0.0809) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, while both the \u003cem\u003eRipk3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and the \u003cem\u003eMlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotype failed to influence the overall survival (OS) of female mice subjected to M/D-driven carcinogenesis (median OS: 125 and 119 days, respectively; \u003cem\u003ep\u003c/em\u003e value: 0.2679 and 0.7347, respectively), the whole-body deletion of \u003cem\u003ePpif\u003c/em\u003e significantly shortened it (median OS: 90 days; \u003cem\u003ep\u003c/em\u003e value: 0.0330), with WT animals exhibiting a median OS of 100 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTaken together, these data indicate that CYPD restrains the natural progression of HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis in female C57BL/6J mice by interfering with early stages of tumorigenesis\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn summary, our data indicate that CYPD \u0026ndash; a fundamental regulator of MPT-driven necrosis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e \u0026ndash; mediates oncosuppressive effects in an immunocompetent mouse model of HR\u003csup\u003e+\u003c/sup\u003e mammary oncogenesis driven by the systemic administration of a chemical carcinogen, \u003cem\u003ei.e.\u003c/em\u003e, DMBA, in the context of supraphysiological PGR signaling, as elicited by slow-release MPA pellets.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e As introduced above, this is a uniquely translational model of HR\u003csup\u003e+\u003c/sup\u003eHER2\u003csup\u003e\u0026minus;\u003c/sup\u003e oncogenesis, as it recapitulates a number of biological, immunological and therapeutic aspects of its human counterpart,\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e hence standing out as a preferential platform for immuno-oncology studies of this specific variant of breast cancer.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Moreover, our findings are fully in line with the well-recognized oncosuppressive role of regulated cell death (RCD) in many of its variants,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e largely (but perhaps not exclusively) reflecting the evolutionary advantage provided to a multicellular organism by signal transduction cascades that coordinate the demise of individual cells bearing excessive macromolecular damage (hence being unable to perform their physiological functions or even at increased risk of malignant transformation) in the context of adequate immunological responses.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCYPD has been shown to mediate various functions that may or may not involve MPT regulation but definitely do not culminate with MPT-driven necrosis, including a paradoxical cytoprotective function in senescent cells.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Thus, it remains possible that the ability of CYPD to suppress HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis in female C57BL/6J mice may be unrelated to RCD via MPT-driven necrosis. This is particularly challenging to formally establish with additional genetic approaches,\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e as most (if not all) proteins that reportedly form or interact with \u0026ndash; hence regulating \u0026ndash; the supramolecular complex responsible for MPT, which is commonly known the permeability transition pore complex (PTPC): (1) exhibit considerable genetic and/or functional redundancy, considerably complicating the implementation of successful knockout strategies \u003cem\u003ein vivo\u003c/em\u003e;\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (2) are critical components of the molecular machinery that ensure mitochondrial ATP synthesis, \u003cem\u003ede facto\u003c/em\u003e being strictly required for survival;\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and (3) at least in some cases, have been conclusively shown to be dispensable for MPT.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Along similar lines, currently available pharmacological inhibitors of the MPT exhibit limited specificity.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e As a standalone example, the pharmacological CYPD inhibitor cyclosporin A (CsA) has major CYPD-independent immunosuppressive effects by inhibiting peptidylprolyl isomerase A (PPIA, best known as CYPA) in T cells.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIntriguingly, CYPD has also been shown to contribute to normal T cell and natural killer (NK) cell functions, at least in preclinical models of infection,\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e raising the possibility that accelerated MPA/DMBA-driven mammary carcinogenesis as observed in \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e C57BL/6J mice may result from defects in natural immunosurveillance.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e We have previously demonstrated that MPA/DMBA-driven mammary tumors develop with an accelerated kinetic in \u003cem\u003eRag2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eIl2rg\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (which lack T cells, B cells and NK cells), as well as in mice receiving an antibody specific for NKG2D (which depletes NK cells and a subpopulation of CD8\u003csup\u003e+\u003c/sup\u003e T cells), but not in \u003cem\u003eRag2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (which lack T and B cells) or in mice receiving CD4- and CD8-targeting antibodies (which are depleted of T cells), globally pointing to NK cells as to central mediators of natural immunosurveillance in this model.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Subjecting C57BL/6J mice to total body irradiation (TBI)-induced myeloablation and reconstituting them with \u003cem\u003ePpif\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e hematopoietic stem cells (and vice versa) will provide additional insights into the role of CYPD expression in radiosensitive vs radioresistant cells in MPA/DMBA-driven mammary carcinogenesis.\u003c/p\u003e \u003cp\u003eDespite this and other open avenues, our findings indicate that CYPD retards HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis in immunocompetent C57BL/6J mice. Of note, CYPD has previously been shown to promote (rather than inhibit) hepatocellular carcinogenesis in mice with non-alcoholic steatohepatitis (NASH)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e as well HR\u003csup\u003e+\u003c/sup\u003eHER2\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis as driven by the MMTV-PyMT construct.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Additional work is hence required to understand whether our data reflect unique immunobiological features of HR\u003csup\u003e+\u003c/sup\u003e breast cancer over other breast cancer subtypes and extramammary neoplasms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions.\u003c/strong\u003e AB performed experimental assessments with help from AS, GP and CG, under supervision from LG. AB and MBV analyzed the data and prepared figures under supervision from LG. LG wrote the manuscript with constructive input from all authors. All authors approve the submitted version of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests.\u003c/strong\u003e LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. All other authors have no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u0026nbsp;\u003c/strong\u003e\u003cem\u003ePpif\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003eRipk3\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eMlkl\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice were a generous gift of Dr. Augustine M.K. Choi (Weill Cornell Medical College, New York, US). The AB lab is/has been supported (as a PI unless otherwise indicated) a Collaborative Research Initiative Grant from the Sandra and Edward Meyer Cancer Center (New York, US) and by startup funds from Fox Chase Cancer Center (Philadelphia, US). The LG lab is/has been supported (as a PI unless otherwise indicated) by one NIH R01 grant (#CA271915), by two Breakthrough Level 2 grants from the US DoD BCRP (#BC180476P1, #BC210945), by a grant from the STARR Cancer Consortium (#I16-0064), by a Transformative Breast Cancer Consortium Grant from the US DoD BCRP (#W81XWH2120034, PI: Formenti), by a U54 grant from NIH/NCI (#CA274291, PI: Deasy, Formenti, Weichselbaum), by the 2019 Laura Ziskin Prize in Translational Research (#ZP-6177, PI: Formenti) from the Stand Up to Cancer (SU2C), by a Mantle Cell Lymphoma Research Initiative (MCL-RI, PI: Chen-Kiang) grant from the Leukemia and Lymphoma Society (LLS), by a Rapid Response Grant from the Functional Genomics Initiative (New York, US), by a pre-SPORE grant (PI: Demaria, Formenti), a Collaborative Research Initiative Grant and a Clinical Trials Innovation Grant from the Sandra and Edward Meyer Cancer Center (New York, US), by startup funds from the Dept. of Radiation Oncology at Weill Cornell Medicine (New York, US), by startup funds from Fox Chase Cancer Center (Philadelphia, US), by industrial collaborations with Lytix Biopharma (Oslo, Norway), Promontory (New York, US) and Onxeo (Paris, France), as well as by donations from Promontory (New York, US), the Luke Heller TECPR2 Foundation (Boston, US), Sotio a.s. (Prague, Czech Republic), Lytix Biopharma (Oslo, Norway), Onxeo (Paris, France), Ricerchiamo (Brescia, Italy), and Noxopharm (Chatswood, Australia).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNewton K, Strasser A, Kayagaki N, Dixit VM. Cell death. Cell 2024, 187(2): 235\u0026ndash;256.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan J, Ofengeim D. A guide to cell death pathways. Nat Rev Mol Cell Biol 2024, 25(5): 379\u0026ndash;395.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKayagaki N, Webster JD, Newton K. Control of Cell Death in Health and Disease. Annu Rev Pathol 2024, 19: 157\u0026ndash;180.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonora M, Giorgi C, Pinton P. Molecular mechanisms and consequences of mitochondrial permeability transition. 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Oncogene 2016, 35(39): 5132\u0026ndash;5143.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cyclosporin A, immunogenic cell death, immunosurveillance, inflammasome, permeability transition pore complex","lastPublishedDoi":"10.21203/rs.3.rs-6265353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6265353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondrial permeability transition (MPT)-driven necrosis and necroptosis are regulated variants of cell death that can drive inflammation or even promote antigen-specific immune responses. In oncological settings, indolent inflammatory reactions have been consistently associated with accelerated disease progression and resistance to treatment. Conversely, adaptive immune responses specific for tumor-associated antigens are generally restraining tumor development and contribute to treatment sensitivity. Here, we harnessed female C57BL/6J mice lacking key regulators of MPT-driven necrosis and necroptosis to investigate whether whole-body defects in these pathways would influence mammary carcinogenesis as driven by subcutaneous slow-release medroxyprogesterone acetate (MPA, M) pellets plus orally administered 7,12-dimethylbenz[\u003cem\u003ea\u003c/em\u003e]anthracene (DMBA, D), an \u003cem\u003ein vivo\u003c/em\u003e model that recapitulates multiple facets of the biology and immunology of human hormone receptor positive (HR\u003csup\u003e+\u003c/sup\u003e) breast cancer. Our data demonstrate that female mice bearing a whole-body, homozygous deletion in peptidylprolyl isomerase F (\u003cem\u003ePpif\u003c/em\u003e), which encodes a key regulator of MPT-driven necrosis commonly known as CYPD, but not female mice with systemic defects in necroptosis as imposed by the whole body-deletion homozygous of receptor-interacting serine-threonine kinase 3 (\u003cem\u003eRipk3\u003c/em\u003e) or mixed lineage kinase domain like pseudokinase (\u003cem\u003eMlkl\u003c/em\u003e), are more susceptible to M/D-driven carcinogenesis than their wild-type counterparts. These findings point to CYPD as to an oncosuppressive protein that restrains HR\u003csup\u003e+\u003c/sup\u003e mammary carcinogenesis in mice, at least potentially via MPT-driven necrosis.\u003c/p\u003e","manuscriptTitle":"CYPD limits HR+ mammary carcinogenesis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 09:19:49","doi":"10.21203/rs.3.rs-6265353/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-03-31T11:33:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-03-28T09:46:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-20T21:24:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-20T15:39:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T11:01:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T02:14:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2025-03-20T02:14:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c851df8-b667-4584-aa0f-65905b3ca086","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46442947,"name":"Biological sciences/Cancer/Breast cancer"},{"id":46442948,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2025-06-11T07:08:39+00:00","versionOfRecord":{"articleIdentity":"rs-6265353","link":"https://doi.org/10.1038/s41420-025-02555-0","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2025-06-10 04:00:00","publishedOnDateReadable":"June 10th, 2025"},"versionCreatedAt":"2025-03-31 09:19:49","video":"","vorDoi":"10.1038/s41420-025-02555-0","vorDoiUrl":"https://doi.org/10.1038/s41420-025-02555-0","workflowStages":[]},"version":"v1","identity":"rs-6265353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6265353","identity":"rs-6265353","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}