Spontaneous Tumors Research Articles

Expression and Function of StAR in Cancerous and Non-Cancerous Human and Mouse Breast Tissues: New Insights into Diagnosis and Treatment of Hormone-Sensitive Breast Cancer.

Breast cancer (BC) is primarily triggered by estrogens, especially 17β-estradiol (E2), which are synthesized by the aromatase enzyme. While all steroid hormones are derived from cholesterol, the rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. Herein, we demonstrate that StAR mRNA expression was aberrantly high in human hormone-dependent BC (MCF7, MDA-MB-361, and T-47D), modest in hormone-independent triple negative BC (TNBC; MDA-MB-468, BT-549, and MDA-MB-231), and had little to none in non-cancerous mammary epithelial (HMEC, MCF10A, and MCF12F) cells. In contrast, these cell lines showed abundant expression of aromatase (CYP19A1) mRNA. Immunofluorescence displayed qualitatively similar patterns of both StAR and aromatase expression in various breast cells. Additionally, three different transgenic (Tg) mouse models of spontaneous breast tumors, i.e., MMTV-Neu, MMTV-HRAS, and MMTV-PyMT, demonstrated markedly higher expression of StAR mRNA/protein in breast tumors than in normal mammary tissue. While breast tumors in these mouse models exhibited higher expression of ERα, ERβ, and PR mRNAs, their levels were undetected in TNBC tumors. Accumulation of E2 in plasma and breast tissues, from MMTV-PyMT and non-cancerous Tg mice, correlated with StAR, but not with aromatase, signifying the importance of StAR in governing E2 biosynthesis in mammary tissue. Treatment with a variety of histone deacetylase inhibitors (HDACIs) in primary cultures of enriched breast tumor epithelial cells, from MMTV-PyMT mice, resulted in suppression of StAR and E2 levels. Importantly, inhibition of StAR, concomitant with E2 synthesis, by various HDACIs, at clinical and preclinical doses, in MCF7 cells, indicated therapeutic relevance of StAR in hormone-dependent BCs. These findings provide insights into the molecular events underlying the differential expression of StAR in human and mouse cancerous and non-cancerous breast cells/tissues, highlighting StAR could serve not only as a novel diagnostic maker but also as a therapeutic target for the most prevalent hormone-sensitive BCs.

C-Myc-IMPDH1/2 axis promotes tumourigenesis by regulating GTP metabolic reprogramming.

Metabolic reprogramming is a hallmark of cancer. Metabolic rate-limiting enzymes and oncogenic c-Myc (Myc) play critical roles in metabolic reprogramming to affect tumourigenesis. However, a systematic assessment of metabolic rate-limiting enzymes and their relationship with Myc in human cancers is lacking. Multiple Pan-cancer datasets were used to develop the transcriptome, genomic alterations, clinical outcomes and Myc correlation landscapes of 168 metabolic rate-limiting enzymes across 20 cancers. Real-time quantitative PCR and immunoblotting were, respectively, used to examine the mRNA and protein of inosine monophosphate dehydrogenase 1 (IMPDH1) in human colorectal cancer (CRC), azoxymethane/dextran sulphate sodium-induced mouse CRC and spontaneous intestinal tumours from APCMin/+ mice. Clone formation, CCK-8 and subcutaneous xenograft model were applied to investigate the possible mechanisms connecting IMPDH1 to CRC growth. Co-immunoprecipitation and protein half-life assay were used to explore the mechanisms underlying the regulation of IMPDH1. We explored the global expression patterns, dysregulation profiles, genomic alterations and clinical relevance of 168 metabolic rate-limiting enzymes across human cancers. Importantly, a series of enzymes were associated with Myc, especially top three upregulated enzymes (TK1, RRM2 and IMPDH1) were positively correlated with Myc in multiple cancers. As a proof-of-concept exemplification, we demonstrated that IMPDH1, a rate-limiting enzyme in GTP biosynthesis, is highly upregulated in CRC and promotes CRC growth in vitro and in vivo. Mechanistically, IMPDH2 stabilizes IMPDH1 by decreasing the polyubiquitination levels of IMPDH1, and Myc promotes the de novo GTP biosynthesis by the transcriptional activation of IMPDH1/2. Finally, we confirmed that the Myc-IMPDH1/2 axis is dysregulated across human cancers. Our study highlights the essential roles of metabolic rate-limiting enzymes in tumourigenesis and their crosstalk with Myc, and the Myc-IMPDH1/2 axis promotes tumourigenesis by altering GTP metabolic reprogramming. Our results propose the inhibition of IMPDH1 as a viable option for cancer treatment.

A Case of Spontaneous Tumor Lysis After Pathologic Femur Fracture in Squamous Cell Lung Cancer.

Tumor lysis syndrome (TLS) occurs when large numbers of tumor cells lyse and release their contents, causing electrolyte derangements and renal injury. It is an uncommon complication of solid tumors and occurs most commonly after cytotoxic therapy rather than spontaneously. In this article, we describe a case of spontaneous TLS in a patient with squamous cell carcinoma of the lung. We also present a literature review of spontaneous TLS occurrence in solid tumors.

Mechanisms of action of vitamin D in delaying aging and preventing disease by inhibiting oxidative stress.

Although several recent studies have shown that vitamin D supplementation beneficially decreases oxidative stress parameters, there is no consensus on this subject in humans. Thus the role of vitamin D supplementation has recently become a controversial topic because large intervention studies in humans have not shown significant benefits. These studies have indicated that supplementation with precursor forms of active vitamin D has no effect on all-cause mortality, cannot reduce the fracture risk of the elderly, cannot reduce the incidence of cancer or cardiovascular disease in the elderly, and cannot significantly reduce the incidence risk of diabetes in the elderly. However, a link between several age-related diseases and enhanced oxidative stress has been found in mice with insufficient or deficient 1,25-dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin D, which indicates that reduced active vitamin D accelerates aging and age-related diseases by increasing oxidative stress. Furthermore, supplementation of exogenous 1,25(OH)2D3, or antioxidants, could dramatically postpone aging, prevent osteoporosis and spontaneous tumor development induced by 1,25(OH)2D insufficiency or deficiency, by inhibiting oxidative stress. Mechanistically, the antioxidative effects of 1,25(OH)2D3 are carried out via the vitamin D receptor (VDR) by activation of the Nrf2 oxidative stress response pathway though transcriptional or posttranscriptional activation of Nrf2 or transcriptional upregulation of Sirt1 and Bmi1 expression. Whether discrepancies between studies in humans and in mice reflect the different forms of vitamin D examined remains to be determined.

The delta subunit of the GABAA receptor is necessary for the GPT2-promoted breast cancer metastasis.

Objectives: Glutamic pyruvate transaminase (GPT2) catalyzes the reversible transamination between alanine and α-ketoglutarate (α-KG) to generate pyruvate and glutamate during cellular glutamine catabolism. The glutamate could be further converted to γ-aminobutyric acid (GABA). However, the role of GPT2 in tumor metastasis remains unclear. Methods: The wound healing and transwell assays were carried out to analyze breast cancer cell migration and invasion in vitro. Gene ontology analysis was utilized following RNA-sequencing to discover the associated molecule function. The mass spectrometry analysis following phosphoprotein enrichment was performed to discover the associated transcription factors. Most importantly, both the tail vein model and Mammary gland conditional Gpt2-/- spontaneous tumor mouse models were used to evaluate the effect of GPT2 on breast cancer metastasis in vivo. Results: GPT2 overexpression increases the content of GABA and promotes breast cancer metastasis by activating GABAA receptors. The delta subunit GABRD is necessary for the GPT2/GABA-induced breast cancer metastasis in xenograft and transgenic mouse models. Gpt2 knockout reduces the lung metastasis of the genetic Gpt2-/- breast cancer in mice and prolongs the overall survival of tumor burden mice. Mechanistically, GPT2-induced GABAA receptor activation increases Ca2+ influx by turning on its associated calcium channel, and the surged intracellular calcium triggers the PKC-CREB pathway activation. The activated transcription factor CREB accelerates breast cancer metastasis by upregulating metastasis-related gene expressions, such as PODXL, MMP3, and MMP9. Conclusion: In summary, this study demonstrates that GPT2 promotes breast cancer metastasis through up-regulated GABA activation of GABAAR-PKC-CREB signaling, suggesting it is a potential target for breast cancer therapy.

Spontaneous tumour lysis syndrome, malignant hypertension and hypercalcemia of malignancy at presentation in an infant with hepatoblastoma

Spontaneous tumour lysis syndrome, malignant hypertension and hypercalcemia of malignancy at presentation in an infant with hepatoblastoma

Incidence of spontaneous tumours in guinea pigs: a retrospective study of 153 cases

The aim of this study was to investigate the incidence of spontaneous tumours in guinea pigs in biopsy and necropsy samples in the monitored period (2016–2022) on a representative number of cases and to determine their occurrence in relation to age and sex of the animals. A total of 204 samples from various pathological lesions were examined, while 153 cases (75.00%) were true tumours and 51 cases (25.00%) were pseudotumorous non-neoplastic lesions. The distribution of the incidence of true neoplasms in relation to sex was uneven. In the examined group, more neoplasms were diagnosed in males (n = 88, 57.52%) compared to females (n = 65, 42.48%). The mean age of guinea pigs with diagnosed tumour in our group was 4.2 years. Thyroid adenocarcinoma was the most frequent tumour. The majority of thyroid tumours (67.86%) were diagnosed in animals aged 4–5 years, as a significant result in terms of incidence in this age range, as well as a predisposition to tumour formation in males (78.57%). Mammary gland tumours occurred in the age range of 2–7 years, with a mean age of incidence of 4.7 years. In all cases, these were malignant variants and showing a significant higher incidence in males, in the age range of 4–5 years. The processing of a representative number of samples and epidemiological data on the incidence of spontaneous tumours in guinea pigs can contribute to the overall expansion of knowledge about this topic in veterinary medicine.

Spontaneous tumour lysis syndrome, malignant hypertension and hypercalcemia of malignancy at presentation in an infant with hepatoblastoma

Spontaneous tumour lysis syndrome, malignant hypertension and hypercalcemia of malignancy at presentation in an infant with hepatoblastoma

Computational systems biology approach for permanent tumor elimination and normal tissue protection using negative biasing: Experimental validation in malignant melanoma as case study.

Complete spontaneous tumor regression (without treatment) is well documented to occur in animals and humans as epidemiological analysis show, whereby the malignancy is permanently eliminated. We have developed a novel computational systems biology model for this unique phenomenon to furnish insight into the possibility of therapeutically replicating such regression processes on tumors clinically, without toxic side effects. We have formulated oncological informatics approach using cell-kinetics coupled differential equations while protecting normal tissue. We investigated three main tumor-lysis components: (ⅰ) DNA blockade factors, (ⅱ) Interleukin-2 (IL-2), and (ⅲ) Cytotoxic T-cells (CD8+ T). We studied the temporal variations of these factors, utilizing preclinical experimental investigations on malignant tumors, using mammalian melanoma microarray and histiocytoma immunochemical assessment. We found that permanent tumor regression can occur by: 1) Negative-Bias shift in population trajectory of tumor cells, eradicating them under first-order asymptotic kinetics, and 2) Temporal alteration in the three antitumor components (DNA replication-blockade, Antitumor T-lymphocyte, IL-2), which are respectively characterized by the following patterns: (a) Unimodal Inverted-U function, (b) Bimodal M-function, (c) Stationary-step function. These provide a time-wise orchestrated tri-phasic cytotoxic profile. We have also elucidated gene-expression levels corresponding to the above three components: (ⅰ) DNA-damage G2/M checkpoint regulation [genes: CDC2-CHEK], (ⅱ) Chemokine signaling: IL-2/15 [genes: IL2RG-IKT3], (ⅲ) T-lymphocyte signaling (genes: TRGV5-CD28). All three components quantitatively followed the same activation profiles predicted by our computational model (Smirnov-Kolmogorov statistical test satisfied, α = 5%). We have shown that the genes CASP7-GZMB are signatures of Negative-bias dynamics, enabling eradication of the residual tumor. Using the negative-biasing principle, we have furnished the dose-time profile of equivalent therapeutic agents (DNA-alkylator, IL-2, T-cell input) so that melanoma tumor may therapeutically undergo permanent extinction by replicating the spontaneous tumor regression dynamics.

Spontaneous Tumor Lysis Syndrome in an Undiagnosed Solid Organ Malignancy: Adenocarcinoma of the Colon

Spontaneous Tumor Lysis Syndrome in an Undiagnosed Solid Organ Malignancy: Adenocarcinoma of the Colon

Superstable homogeneous lipiodol-ICG formulation: initial feasibility and first-in-human clinical application for ruptured hepatocellular carcinoma.

The most common treatment of spontaneous tumor rupture hemorrhage (STRH) is transcatheter arterial embolization (TAE) followed by liver resection, and surgical navigation using near-infrared fluorescence is effective method for detecting hidden lesions and ill-defined tumor boundaries. However, due to the blockage of the tumor-supplying artery after effective TAE treatment, it is difficult to deliver sufficient fluorescent probes to the tumor region. In this study, we report on the successful application of superstable homogeneous intermixed formulation technology (SHIFT) in precise conversion hepatectomy for ruptured hepatocellular carcinoma (HCC). A homogeneous lipiodol-ICG formulation obtained by SHIFT (SHIFT-ICG) was developed for clinical practice for STRH. A ruptured HCC patient received the combined protocol for embolization and fluorescence surgical navigation and exhibited excellent hemostatic effect. Lipiodol and ICG were both effectively deposited in the primary lesion, including a small metastatic lesion. In follow-up laparoscopic hepatectomy, SHIFT-ICG could clearly and precisely image the full tumor regions and boundaries in real time, and even indistinguishable satellite lesions still expressed a remarkable fluorescence intensity. In conclusion, the simple and green SHIFT-ICG formulation can be effectively used in emergency embolization hemostasis and later precise fluorescence navigation hepatectomy in patients with ruptured HCC bleeding and has high clinical application value.

Identification of SNPs and InDels probably associated with the development of spontaneous tumors in radish (<i>Raphanus sativus</i> L.)

Radish (Raphanus sativus L.) is an agronomically important root crop belonging to the Brassicaceae family. A genetic collection of inbred radish lines, which was based on contrasting genetically determined traits including trait ability to develop tumors, was created at St. Petersburg State University in the middle of the 20th century [1]. Full genetics network controlling this trait still remains unclear [2], and elucidation of its mechanisms can help reveal the key regulators of systemic mechanisms that control cell proliferation and differentiation.
 The purpose of this work is to identify single nucleotide polymorphisms (SNPs) and InDels in genes which are candidates for participation in the tumor development process within in the tumor-forming radish line compared to the non-tumor radish line.
 We have assembled the genomes of two lines contrasting in the ability to tumorigenesis, annotated them, aligned sequences per assembly, identified candidate genes and differences in the structure of these genes in contrasting radish lines using bioinformatics tools. Bioinformatics data were confirmed by the sequencing by Sanger method.
 As a result, in the tumor-forming radish line we identified 151 genes with InDels in their coding regions, which led to various variants of frameshift. Moreover, we detected 39 genes with single nucleotide substitutions (SNPs). According to the gene pathway enrichment analysis, the corresponding genes were classified into the several groups.
 Thus, the data obtained will allow us to clarify in more detail the genetic control mechanisms of the tumor development in radish.
 The work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with the agreement No. 075-15-2022-322.

Targeting SLP2-mediated lipid metabolism reprograming restricts proliferation and metastasis of hepatocellular carcinoma and promotes sensitivity to Lenvatinib.

SLP2, a protein located on mitochondrial, has been shown to be associated with mitochondrial biosynthesis. Here we explored the potential mechanisms by which SLP2 regulates the development of hepatocellular carcinoma. SLP2 could bind to the c-terminal of JNK2 to affect the ubiquitinated proteasomal degradation pathway of JNK2 and maintain the protein stability of JNK2. The increase of JNK2 markedly increases SREBP1 activity, promoting SREBP1 translocation into the nucleus to promote de novo lipogenesis. Alteration of the JNK2 C-terminal disables SLP2 from mediating SLP2-enhanced de novo lipogenesis. YTHDF1 interacts with SLP2 mRNA in a METTL3/m6A-dependent manner. In a spontaneous HCC animal model, SLP2/c-Myc/sgP53 increases the incidence rate of spontaneous HCC, tumor volume, and tumor number. Importantly, statistical analyses show that levels of SLP2 correlate with tumor sizes, tumor metastasis, overall survival, and disease-free survival of the patients. Targeting the SLP2/SREBP1 pathway effectively inhibits proliferation and metastasis of HCC tumors with high SLP2 expression in vivo combined with lenvatinib. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic SLP2, providing a mechanistic link between de novo lipogenesis and HCC.

Dexmedetomidine provides type-specific tumour suppression without tumour-enhancing effects in syngeneic murine models

BackgroundDexmedetomidine is a widely used anaesthetic adjuvant for cancer resection surgeries. However, recent reports suggest that it may promote tumour growth or metastasis, so it is essential to clarify its tumour-related effects. MethodsSeven syngeneic murine tumour models were used to assess the impact of dexmedetomidine on primary tumour growth, spontaneous tumour metastasis, and surgical resection-associated metastasis. Cancer cell proliferation and apoptosis experiments, terminal deoxynucleotidyl transferase dUTP nick-end labelling assays, immune cell analysis, specific T-cell depletion experiments, and gene transcription analysis were conducted to identify the underlying mechanisms. ResultsDexmedetomidine did not affect growth of EO771 or 4T1 breast tumours, LAP0297 or LLC lung tumours, MCA205 fibrosarcoma, or their spontaneous lung metastases. It did not promote lung metastasis after breast cancer resection. Dexmedetomidine significantly suppressed MCA38 and CT26 colorectal tumour growth (P<0.01) and promoted apoptosis in MCA38 tumour tissues (P<0.05) without affecting proliferation and apoptosis of MCA38 tumour cells in vitro, suggesting indirect anti-tumour effects. Dexmedetomidine increased the proportions of intratumour CD4+ T (P<0.01), CD8+ T (P<0.001), and natural killer cells (P<0.01), and it upregulated transcription of the cytotoxicity-related genes Infg, Tnfa, and Cxcl9 (P<0.05) in MCA38 tumours. Either CD8+ or CD4+ T-cell depletion reversed the anti-tumour effects of dexmedetomidine on MCA38 tumours (P<0.05). ConclusionsDexmedetomidine conferred colorectal tumour-type specific suppression by modulation of tumour CD4+ and CD8+ T cells without tumour-enhancing effects.

The Microtubule Destabilizer Eribulin Synergizes with STING Agonists to Promote Antitumor Efficacy in Triple-Negative Breast Cancer Models.

Eribulin is a microtubule destabilizer used in the treatment of triple-negative breast cancer (TNBC). Eribulin and other microtubule targeted drugs, such as the taxanes, have shared antimitotic effects, but differ in their mechanism of microtubule disruption, leading to diverse effects on cellular signaling and trafficking. Herein, we demonstrate that eribulin is unique from paclitaxel in its ability to enhance expression of the immunogenic cytokine interferon beta (IFNβ) in combination with STING agonists in both immune cells and TNBC models, including profound synergism with ADU-S100 and E7766, which are currently undergoing clinical trials. The mechanism by which eribulin enhances STING signaling is downstream of microtubule disruption and independent of the eribulin-dependent release of mitochondrial DNA. Eribulin did not override the requirement of ER exit for STING activation and did not inhibit subsequent STING degradation; however, eribulin significantly enhanced IRF3 phosphorylation and IFNβ production downstream of the RNA sensing pathway that converges on this transcription factor. Additionally, we found that eribulin enhanced the population of activated CD4+ T-cells in vivo when combined with either a STING agonist or tumor, demonstrating the ability to function as an immune adjuvant. We further interrogated the combination of eribulin with ADU-S100 in the MMTV-PyVT spontaneous murine mammary tumor model where we observed significant antitumor efficacy with combination treatment. Together, our findings demonstrate that microtubule targeted chemotherapeutics have distinct immunological effects and that eribulin's ability to enhance innate immune sensing pathways supports its use in combination with immunotherapies, such as STING agonists, for the more effective treatment of TNBC and other malignancies.

Abstract B009: A SOX9-B7x axis safeguards dedifferentiated tumor cells from immunosurveillance to enable DCIS progression

Abstract Accumulating evidence has demonstrated a central role of dedifferentiation in carcinogenesis. In basal-like breast cancer mouse models, upregulation of SOX9 drives dedifferentiation of luminal progenitors to cells with luminal-basal hybrid features resembling embryonic multipotent mammary stem cells. Additionally, genetic inactivation of SOX9 inhibits dedifferentiation and blocks the progression of in situ tumors to malignant carcinoma. Whether the host immune system can recognize these dedifferentiated cells and how they eventually evade immunosurveillance are unclear. Here, we showed that SOX9 was necessary for limiting infiltrating T lymphocytes to protect dedifferentiated cells from immune surveillance in premalignant lesions of basal-like breast cancer. SOX9 deletion in spontaneous BLBC tumor models caused massive T lymphocyte infiltration and elimination of dedifferentiated cells. Antibody-mediated T cell depletion rescued the accumulation of dedifferentiated tumor cells in SOX9-deficient DCIS and its progression to invasive tumors. Mechanistically, through both stimulation of the STAT3 pathway and direct transcriptional activation, SOX9 upregulated the expression of the immune checkpoint B7x, which mediated the action of SOX9 in immunosuppression. In advanced tumors, B7x blockade reactivated antitumor immunity and effectively inhibited tumor growth. Furthermore, B7x depletion overcame the resistance to anti-PD-L1 immunotherapy. Collectively, these findings uncovered a dedifferentiation-associated immune evasion mechanism and demonstrated the therapeutic potential of targeting the SOX9-B7x pathway to eradicate dedifferentiated tumor cells for intercepting breast cancer progression. Citation Format: Yu Liu, Peter John, John R Christin, Maja Oktay, XingXing Zang, Wenjun Guo. A SOX9-B7x axis safeguards dedifferentiated tumor cells from immunosurveillance to enable DCIS progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B009.

Abstract B032: Native sgRNA capture and sequencing (NSC-seq) reveals tumor formation in ApcMin/+ mice and in human colorectal cancer is an oligoclonal process

Abstract CRISPR-Cas9-based technologies have been developed to address a wide range of biological questions, including molecular recording of embryonic development, genetic screening, and tracking tumor evolution at the single-cell resolution. We developed a single-cell lineage tracking platform called native sgRNA Capture and sequencing (NSC-seq) to leverage CRISPR technology for clonal analysis of intestinal tumors in the ApcMin/+ mouse model. We combined CRISPR-induced mutations with spontaneously accumulating mitochondrial variants to enable highly efficient lineage-tracking of single cells. We generated lineage-tracked ApcMin/+ tumors and found that there are more than two independently arising clones within a tumor. We also found oligoclonal tumors within human colorectal specimens using this technology, supporting the oligoclonality of intestinal tumor development across species. Finally, we demonstrated the application of this technology in understanding the rate of loss-of-heterozygosity events in Apc as related to the cellular turnover rates of different intestinal regions. Overall, NSC-seq enables in vivo molecular recording of spontaneous intestinal tumor development at single-cell resolution. Citation Format: Mirazul Islam, Yilin Yang, Vishal M. Shah, Alan J. Simmons, Yanwen Xu, Robert J. Coffey, Ken S. Lau. Native sgRNA capture and sequencing (NSC-seq) reveals tumor formation in ApcMin/+ mice and in human colorectal cancer is an oligoclonal process [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B032.

EXTRACELLULAR MATRIX OF EXPERIMENTAL HEPATOCARCINOMA G-29 UNDER THE INFLUENCE OF LITHIUM CARBONATE

Hepatocellular carcinoma (HCC) is one of the most aggressive human tumors with a high mortality rate. Despite the fact that the incidence rate of this sort of tumors is high, the mechanisms of its pathogenesis and metastasis are not fully understood, and the existing therapy is often unsuccessful. There is evidence in the reference literature available that in patients with HCC, alterations occur in the microenvironment of the tumor tissue, including the extracellular matrix (ECM) that stimulates the growth and metastasis of the HCC cells. Therefore, the aim of our study was to investigate the state of the tumor ECM during the spontaneous tumor development and with lithium carbonate introduction.

Immunogenic Death of Hepatocellular Carcinoma Cells in Mice Expressing Caspase-Resistant ROCK1 Is Not Replicated by ROCK Inhibitors

The morphological changes during apoptosis help facilitate "immunologically silent" cell death. Caspase cleavage of the ROCK1 kinase results in its activation, which drives the forceful contraction of apoptotic cells. We previously showed that when ROCK1 was mutated to render it caspase-resistant, there was greater liver damage and neutrophil recruitment after treatment with the hepatotoxin diethylnitrosamine (DEN). We now show that acute DEN-induced liver damage induced higher levels of pro-inflammatory cytokines/chemokines, indicative of immunogenic cell death (ICD), in mice expressing non-cleavable ROCK1 (ROCK1nc). Hepatocellular carcinoma (HCC) tumours in ROCK1nc mice had more neutrophils and CD8+ T cells relative to mice expressing wild-type ROCK1, indicating that spontaneous tumour cell death also was more immunogenic. Since ICD induction has been proposed to be tumour-suppressive, the effects of two distinct ROCK inhibitors on HCC tumours was examined. Both fasudil and AT13148 significantly decreased tumour numbers, areas and volumes, but neither resulted in greater numbers of neutrophils or CD8+ T cells to be recruited. In the context of acute DEN-induced liver damage, AT13148 inhibited the recruitment of dendritic, natural killer and CD8+ T cells to livers. These observations indicate that there is an important role for ROCK1 cleavage to limit immunogenic cell death, which was not replicated by systemic ROCK inhibitor administration. As a result, concomitant administration of ROCK inhibitors with cancer therapeutics would be unlikely to result in therapeutic benefit by inducing ICD to increase anti-tumour immune responses.

Aberrant expression and localization of the RAP1 shelterin protein contribute to age-related phenotypes.

Short telomeres induce a DNA damage response (DDR) that evokes apoptosis and senescence in human cells. An extant question is the contribution of telomere dysfunction-induced DDR to the phenotypes observed in aging and telomere biology disorders. One candidate is RAP1, a telomere-associated protein that also controls transcription at extratelomeric regions. To distinguish these roles, we generated a knockin mouse carrying a mutated Rap1, which was incapable of binding telomeres and did not result in eroded telomeres or a DDR. Primary Rap1 knockin embryonic fibroblasts showed decreased RAP1 expression and re-localization away from telomeres, with an increased cytosolic distribution akin to that observed in human fibroblasts undergoing telomere erosion. Rap1 knockin mice were viable, but exhibited transcriptomic alterations, proinflammatory cytokine/chemokine signaling, reduced lifespan, and decreased healthspan with increased body weight/fasting blood glucose levels, spontaneous tumor incidence, and behavioral deficits. Taken together, our data present mechanisms distinct from telomere-induced DDR that underlie age-related phenotypes.