This file contains 6 supplementary tables referred to in the text of the manuscript

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Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. The cleavage of full-length cyclin E (FL-cycE) to low-molecular weight isoforms (LMW-E) dramatically alters substrate specificity, promoting G1-S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. In this study, analysis of tumor samples of patients with TNBC revealed that coexpression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared with FL-cycE, LMW-E specifically upregulates PKMYT1 expression and protein stability, thereby increasing CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E-dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breakage, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and patient-derived xenografts of LMW-E-high TNBC but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype. Significance: PKMYT1 upregulation and CDK1 phosphorylation in triple-negative breast cancer expressing low-molecular weight cyclin E leads to suboptimal responses to chemotherapy but sensitizes tumors to PKMYT1 inhibitors, proposing a personalized treatment strategy.

How cells coordinate cell cycling with cell survival and death remains incompletely understood. Here, we show that cell cycle arrest has a potent suppressive effect on ferroptosis, a form of regulated cell death induced by overwhelming lipid peroxidation at cellular membranes. Mechanistically, cell cycle arrest induces diacylglycerol acyltransferase (DGAT)–dependent lipid droplet formation to sequester excessive polyunsaturated fatty acids (PUFAs) that accumulate in arrested cells in triacylglycerols (TAGs), resulting in ferroptosis suppression. Consequently, DGAT inhibition orchestrates a reshuffling of PUFAs from TAGs to phospholipids and re-sensitizes arrested cells to ferroptosis. We show that some slow-cycling antimitotic drug–resistant cancer cells, such as 5-fluorouracil–resistant cells, have accumulation of lipid droplets and that combined treatment with ferroptosis inducers and DGAT inhibitors effectively suppresses the growth of 5-fluorouracil–resistant tumors by inducing ferroptosis. Together, these results reveal a role for cell cycle arrest in driving ferroptosis resistance and suggest a ferroptosis-inducing therapeutic strategy to target slow-cycling therapy-resistant cancers.

Abstract Background: Cyclin-dependent-kinase-4/6 inhibitors (CDK4/6is) plus endocrine therapy (ET) are standard of care first-line treatment for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (mBC). However, the emergence of resistance to CDK4/6is plus ET presents a clinical challenge with few treatment alternatives. The effectiveness of CDK4/6is in patients with triple-negative (TN) breast cancer remains uncertain, although the potential synergy with other targeted therapies is currently under investigation. Our study demonstrates the enhanced and synergistic activity of BLU-222, a selective CDK2 inhibitor, when combined with CDK4/6is in preclinical models of both HR+/HER2- and TN breast cancer resistant to CDK4/6is. Methods: Palbociclib resistant (PR) HR+/HER2- (MCF7 and T47D) and TN (MDA-MB-231 and BT-20) breast cancer cell lines were generated by escalating palbociclib concentrations in culture. Using SynergyFinder, we assessed the effect of BLU-222 alone and in combination with palbociclib in the highest single-agent model in vitro. The effectiveness of BLU-222, alone or in combination with palbociclib, was further evaluated in four patient-derived xenograft (PDX) models from HR+/HER2- patients whose tumors progressed after palbociclib plus ET, two TN PDX models, and a TN breast cancer transgenic model driven by tumor-specific forms of cyclin E. Results: The PR HR+/HER2- and TN breast cancer cell lines, unresponsive to palbociclib, exhibited significantly increased sensitivity to BLU-222. The combination of BLU-222 and palbociclib in all four cell lines revealed a robust synergistic effect in PR cells, inducing enhanced apoptosis and cell cycle alterations in G1 or G2/M phases. Treatment with BLU-222 and palbociclib demonstrated substantial antitumor activity in all six PDX models and the cyclin E high TN transgenic models, surpassing the effects of individual treatments in each model. This combination led to lasting tumor regression and extended survival, even after treatment discontinuation. Mechanistically, treatment with BLU-222, alone or combined with palbociclib, induced the expression of p21 and/or p27 in all in vivo models, which we hypothesize sensitizes tumors to palbociclib. In vitro, the CRISPR knockout of p21 or p27 in MCF-7 PR cells abolished the synergistic activity of BLU-222 and palbociclib, confirming the crucial involvement of p21 and p27 in influencing the treatment's effectiveness. Conclusions: We found robust activity of the CDK2 inhibitor, BLU-222 when combined with CDK4/6is in resistant HR+/HER2- and TN breast cancer cell lines and in vivo models. These results support the potential clinical utility of BLU-222 in combination with CDK4/6is for the treatment of both subtypes of breast cancer. Citation Format: Linjie Luo, Yan Wang, Tuyen Bui, Mi Li, Serena Kim, Juliana Navarro-Yepes, Nicole M. Kettner, Debasish Tripathy, Kelly K. Hunt, Kerrie Faia, Khandan Keyomarsi. Anti-tumor activity of CDK2 inhibitor BLU-222 in combination with CDK4/6 inhibitors for overcoming resistance in HR positive and triple negative metastatic breast cancers models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4622.

Abstract Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. Significance: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.

Background: Cyclin E is post-translationally modified by neutrophil elastase mediated proteolytic cleavage to generate the low molecular weight isoforms of cyclin E (LMW-E) that are detected in various human cancers. We previously reported that 70% of triple negative breast cancers (TNBC) examined overexpress LMW-E, and these patients have a poor prognosis. Expression of LMW-E promotes genomic instability by causing DNA replication stress. PKMYT1 prevents premature mitotic entry by catalyzing CDK1 phosphorylation at T14, essential for preventing DNA damage and cell death when cyclin E, including LMW-E, is overexpressed. In this study, we tested the hypothesis that LMW-E positive status can be used as a biomarker of response in selecting TNBC patients who are likely to respond to RP-6306, a first in-class and selective inhibitor of PKMYT1 kinase. Results: Assessment of pre-treatment breast biopsies from TNBC patients (n=40) enrolled in a neoadjuvant chemotherapy prospective study for LMW-E and CDK1-pT14 revealed significant positive correlation between these two proteins. Furthermore, positivity of both biomarkers was associated with lack of pathologic complete response (pCR) to neoadjuvant chemotherapy. We next examined the mechanism of response to RP-6306 in vitro and in vivo using TNBC cell lines, patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models expressing human LMW-E (hLMW-E). In vitro results using 7 different TNBC cell lines, revealed that high LMW-E levels are significantly predictive of response to RP-6306 (R2=0.78, p= 0.008), while LMW-E knockdown resulted in a 7X increase in IC50 values of RP-6306 (p<0.001). In high LMW-E cells, treatment with RP-6306 resulted in significant (i) downregulation of CDK1-pT14, PKMYT1, WEE1, cyclin B and pRb, (ii) accumulation of sub-G1 and polyploid cell population, (iii) apoptosis, (iv) accumulation of chromosomal breakage, (v) increased DNA damage (increase in γ-H2AX and 53BP1 foci/cell) and lack of DNA repair (downregulation of Rad51), and (vi) premature mitotic entry. Treatment of both breast cancer PDX models and hLMW-E transgenic tumors with RP-6306 revealed that only in animals with high LMW-E tumors, treatment results in significant decrease in tumor volume. However, RP-6306 was ineffective in reducing tumor volume in low cyclin E in vivo models. Immunohistochemical analysis revealed that in vivo efficacy of RP-6306 (in both PDX and transgenic models) was concomitant with increase in γ-H2AX and decrease in CDK1-pT14 and Ki67. Conclusion: Collectively, our results show that overexpression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306. Mechanistically, LMW-E overexpressing TNBC cells activate CDK1 (in vitro and in vivo) to accelerate premature mitotic entry, leading to DNA damage and apoptosis. Citation Format: Amriti Lulla, Tuyen D. Nguyen, Mi Li, Sofia Mastoraki, Yan Wang, Tuyen Bui, Marc Pina, Spiridon Tsavachidis, Gary Marshall, Kelly K. Hunt, Khandan Keyomarsi. Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 950.

Radiation-induced oral mucositis is the most common complication for patients who receive head/neck radiotherapy. Nicotinamide adenine dinucleotide (NAD+) is vital for DNA damage repair under ionizing radiation, through functioning as either the substrate for protein poly(ADP-ribosyl)ation at DNA break sites or the cofactor for multiple DNA repair-related enzymes, which therefore can result in a significant consumption of cellular NAD+ during DNA repair. Mammalian cells produce NAD+ mainly by recycling nicotinamide via the salvage pathway, in which the rate-limiting step is governed by nicotinamide phosphoribosyltransferase (NAMPT). However, whether NAMPT is co-opted under ionizing radiation to timely fine-tune NAD+ homeostasis remains elusive. Here we show that ionizing radiation evokes NAMPT activation within 30 min without apparent changes in its protein expression. AMPK rapidly phosphorylates NAMPT at S314 under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing NAMPT binding with its substrate phosphoribosyl pyrophosphate (PRPP). AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD+ level in the irradiated cells and facilitates DNA repair and cell viability. Our findings demonstrate a new post-translational modification-based signalling route, by which cells can rapidly orchestrate NAD+ metabolism to support DNA repair, thereby highlighting NAMPT as a potential target for the prevention of ionizing radiation-induced injuries.

Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.

Low Molecular Weight Cyclin E (LMW-E) are the tumor specific, oncogenic forms of cyclin E that are post translationally generated by neutrophil elastase (NE) mediated cleavage of the 50 KDa full-length cyclin E1 (FL-cycE, encoded by CCNE1 gene). While FL-cycE localizes mainly to the cell nucleus, LMW-E lack the N-terminus nuclear localization signal and are detected in both the nucleus and cytoplasm. Compared to FL-cycE, LMW-E exhibit longer half-life and higher affinity to their kinase partner CDK2 and are resistant to natural CDK inhibitors such as p21 and p27. It is currently assumed that LMW-E drive the tumorigenic process by promoting G1/S cell cycle transition and accelerating mitotic exit. Here we report that LMW-E overexpression also promotes genomic instability by deregulating DNA replication in a CDC6 dependent manner. To this end, we developed an immunohistochemistry (IHC) assay with a cyclin E antibody that can identify LMW-E expressing tumors and examined the association of genetic instability of each tumor with LMW-E status in 2 different cohorts of breast cancer patients. Cohort 1 is a retrospective cohort of 725 patients with stage I-II breast cancer treated at MD Anderson (Houston, TX) between 1985 and 1999. Cohort 2 is a prospective cohort of 85 patients with stage I-II breast cancer who enrolled in our study at MD Anderson between January 2000 and June 2010. Our results show that positive LMW-E status in stage 1 or 2 breast cancer patients correlates with increasing copy number variations, as identified by Molecular Inversion Probe (MIP) in cohort 1 and somatic mutations, as identified by Whole Exome Sequencing (WES) in cohort 2. Second, using immortalized human mammary epithelial cells (hMECs) engineered to express doxycycline inducible LMW-E or FL-cycE in CCNE1 knock-out background, we found that FL-cycE over-expression leads to DNA damage, cell cycle arrest and cell death. LMW-E overexpression, on the other hand, facilitates cell proliferation with damaged DNA, resulting in multi-nuclei and micro-nuclei formation in daughter cells. Third, overexpression of FL-cycE reduces chromatin bound MCM complex, while LMW-E overexpression promotes the chromatin loading of pre-replication complex including MCMs. Lastly, we show that LMW-E but not FL-cycE is the major form of cyclin E that binds to the chromatin. Specifically, in both LMW-Einducible hMECs and LMW-Ehighbreast tumor cell lines, CDC6 is required for the nuclear translocation and chromatin loading of LMW-E. Our findings have revealed the unique oncogenic function of LMW-E in deregulating replication licensing, promoting replication stress tolerance and genomic instability that fuels tumor development. These findings also provide potential novel therapeutic strategies for treating LMW-Ehigh breast tumors, who do not respond to the current standard of care therapies. Citation Format: Mi Li, Kwang Huei Low, Tuyen Bui, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E facilitate replication stress tolerance in breast cancer development [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-05-02.

Purpose: Low Molecular Weight Cyclin E (LMW-E) are the oncogenic forms of cyclin E and were originally discovered in breast cancer (BC). LMW-E are generated from N-terminal cleavage of the 50 KDa, full-length cyclin E1 (FL-cycE). Our laboratory has established that LMW-E expression (i) correlates with poorer survival in BC patients; (ii) increases in frequency as BC progress from ductal carcinoma in situ to invasive ductal carcinoma; (iii) drives spontaneous and metastatic BC in murine transgenic models; (iii) causes human mammary epithelial cells (hMECs) to transform in vitro and form tumors in vivo. Elucidating the critical oncogenic functions and essential downstream factors of LMW-E is a current void in the field and may have important therapeutic implications. Experimental Design: Immunohistochemistry (IHC) analysis were performed using tumor specimens (n=725) from breast cancer patients diagnosed with Stage 1 or 2 disease to determine the level of LMW-E. Micro-dissected tumor DNA from these tissues were analyzed for copy number variations (CNVs) using Molecular Inversion Probe (MIP) based arrays. CCNE1(encoding Cyclin E1) knock-out 76NE6 and 76NF2V hMEC lines were generated by CRISPR, followed by transfection of lentivirus vector expressing doxycycline inducible EGFP fused LMW-E or FL-cycE. Time-lapse live cell imaging was performed to monitor the cell growth and phenotypes after induced expression of LWM-E or FL-cycE. DNA replication and replication stress were examined by BrdU labeling and immunofluorescent (IF) assays using antibodies against BrdU and Replication Protein A (RPA). IF for gamma-H2AX and western blotting for phosphor-RPA32, ATR-CHK1 and ATM-CHK2 pathways were performed to determine the DNA damage and responses. Results: Analysis of CNVs for the 725 tumors stratified by the cyclin E phenotypes reveal that the frequency of the CNVs (gains & losses) were the most significant in patients whose tumors expressed LMW-E and were predictive of poor prognosis independent of BC subtypes. DNA damage signals including gamma-H2AX foci and phosphor-CHK1 were similarly enhanced in hMECs induced for FL-cycE or LMW-E over-expression. Different effects of FL-cycE and LMW-E on DNA replication and replication stress were observed. Induced FL-cycE overexpression attenuated increased RPA foci and RPA phosphorylation, inhibits DNA replication and cell growth, and ultimately lead to cell death. LMW-E overexpression facilitated DNA replication and cell proliferation with damaged DNA, resulting in multi-nuclei and micro-nuclei formation in daughter cells. Conclusion: LMW-E expression positively correlates with genomic instability in BC samples. LMW-E facilitates hMECs to by-pass replication stress induced tumor barrier and survive with damaged DNA, resulting in abnormal nuclei formation, an oncogenic phenotype. Citation Format: Mi Li, Kwang Huei Low, Tuyen Bui, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E facilitates replication stress tolerance in breast cancer development [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-04.

BACKGROUNDS/AIMS:Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to mammals that produce vitamin C in their liver. Gulo serves as a crucial enzyme for vitamin C synthesis in mammals, but the characteristics and localization of its homologous genes during kidney development in Xenopus laevis, an amphibian, remains unknown.METHODS:We aligned amino acid sequences of Gulo across different species by using bioinformatics methods and detected patterns of expression for Gulo during kidney development by using RT-PCR and in situ hybridization.RESULTS:We identified a new site on the X. laevis genome, LOC495407. Sequence alignment analysis indicated this fragment is highly conserved and homologous to gulo genes in mammals. RT-PCR and in situ hybridization results reveal that X. laevis gulo is maternally expressed during the early stages of embryonic development, particularly, in the tubules of the pronephros from the middle tail-bud stage and onward in embryos.CONCLUSION:Gulo is a novel specific marker for pronephros tubules in X. laevis, and may be used as a potential marker for kidney development studies and disease diagnosis in mammals.

ABSTRACT Virus infection triggers immediate innate immune responses. Apoptosis represents another effective means to restrict virus invasion, besides robust expression of host cytokines and chemokines. IRF3 was recently demonstrated to be indispensable for Sendai virus (SeV)-induced apoptosis, but the underlying mechanism is not fully understood. Here we report that a dynamic protein complex, Tom70/Hsp90/IRF3/Bax, mediates SeV-induced apoptosis. The cytosolic proapoptotic protein Bax interacts specifically with IRF3 upon virus infection. The mitochondrial outer membrane protein Tom70 recruits IRF3 to mitochondria via Hsp90. Consequently, the relocation of Bax onto mitochondria induces the leakage of cytochrome c into the cytosol and initiates the corresponding apoptosis. Interestingly, IKK-i is essential for this apoptosis, whereas TBK1 is dispensable. Collectively, our study characterizes a novel protein complex that is important for SeV-induced apoptosis. IMPORTANCE Apoptosis is an effective means of sacrificing virus-infected cells and restraining the spread of virus. In this study, we demonstrate that IRF3 associates with Bax upon virus infection. Tom70 recruits this protein complex to the mitochondrial outer membrane through Hsp90, which thus induces the release of cytochrome c into the cytosol, initiating virus-induced apoptosis. Interestingly, IKK-i plays an essential role in this activation. This study uncovers a novel mechanism of SeV-induced apoptosis.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas accounting for 3%-10% of all soft tissue sarcomas. Neurofibromatosis type 1 (NF1) is the most important known risk factor. MPNSTs are often diagnosed at an advanced stage when distant metastases have developed. Although surgical resection remains the main treatment for MPNSTs, complete surgical resection is rarely possible. The prognosis for patients with MPNSTs is poor. There is an urgent need for improved therapies. To this end, we investigated whether microRNA (miR), specifically miR-204, might be implicated in MPNSTs because it is located at a cancer-associated genomic region exhibiting high frequency of loss of heterozygosity in tumors. We show that miR-204 expression is downregulated in NF1 and non-NF1 MPNST tumor tissues and in tumor cell lines. Restoring miR-204 expression in MPNST cell lines STS26T (non-NF1), ST88-14 (NF1), and T265p21 (NF1) significantly reduces cellular proliferation, migration, and invasion in vitro. Restoring miR-204 expression in STS26T decreases tumor growth and malignant progression in vivo. We also report that miR-204 inhibits Ras signaling and expression of high mobility group gene A2. These findings support the hypothesis that miR-204 plays critical roles in MPNST development and tumor progression. miR-204 may represent a novel biomarker for diagnosis and a candidate target with which to develop effective therapies for MPNSTs.

UNLABELLED:OBJECTIVE To summary the functional roles and molecular mechanisms of microRNA (miRNA) in osteoblast differentiation so as to supply information for basic and clinical researches.METHODS:Recent literature concerning miRNA in osteoblast differentiation was reviewed. The information was classified and summarized.RESULTS:miRNAs critically regulate bone morphogenetic protein, transforming growth factor beta, and Wnt/beta-catenin signaling pathways during osteoblast differentiation. In pathological conditions, especially in some disorders of abnormal osteoblast differentiation, down-regulated miRNA expression has been observed.CONCLUSION:miRNA may represent a novel biomarker for diagnosis, and a candidate target therapies for the disorders with abnormal osteoblast differentiation.

Metastases account for 90% of lung cancer mortalities, frequently target the skeleton and lead to rapid deterioration in quality of life. The molecular mechanism underlying bone metastases is largely unknown. Development of xenograft mouse models, such as the severe combined immunodeficient (SCID) CB-17 mouse and the non-obese diabetic (NOD)/SCID mouse, both of which lack functional B- and T-cells and are able to host allogeneic or xenogeneic tumor cells, has made great contributions in this area. However, residual natural killer (NK) cells in these models are able to significantly modify local tumor growth and metastasis. Treatment with anti-murine IL-2 receptor β chain Ab (TM-β1) antibody can abrogate NK cell activity in vivo; however, the antibody treatment may result in unexpected effects and the stability is hard to control. To overcome these shortcomings, we evaluated xenografts in NOD-scid IL2Rγ(null) immunodeficient mice that lacked mature T cells, B cells and functional NK cells. We compared the target tissue distribution of the human small cell lung cancer cell lines SBC-5 and SBC-3. Gross necropsy and whole skeletal X-ray film examination of the host mice were conducted 30 days post-tail vein injection. The SBC-5 cells colonized bone and formed lytic lesions. The cells also colonized liver, spleen and, less frequently, the pancreas, ovary and kidney. The SBC-3 cell xenografts formed easily visible tumor foci in the liver, pancreas, ovary/uterus and kidney, but not bone metastases. Our results showed that SBC-5 cells in NOD-scid IL2Rγ(null) immunodeficient mice provide a suitable xenograft model system for bone metastasis of human lung cancer. This novel animal model may therefore be used to study the molecular pathway of bone metastases and to evaluate targets for effective therapies.

SHARPIN is a novel protein thought to interact with SHANK family and is widely expressed in multiple tissues/cells, including osteoblasts and osteoclasts. Loss-of-function of Sharpin develops the chronic proliferative dermatitis mutation (CPDM) in mice as well as a severe inflammation in other organs. The actual function of SHARPIN is poorly understood. Our aim was to determine the functional roles of SHARPIN in bone metabolism by using CPDM mice. The skeletal phenotypes were determined by peripheral quantitative computed tomography, micro-computed tomography, and quantitative real-time RT-PCR, the cellular functions of osteoblasts and osteoclasts were investigated by ex vivo cell culture. Compared to wild-type controls, CPDM mice demonstrated significantly lower total and cortical bone mineral content and bone mineral density, trabecular and cortical bone volume, and trabecular number. The mRNA expression of Runx2, osterix, type I collagen, and osteocalcin was significantly lower in the bone from CPDM mice. Osteoclasts and osteoblasts from CPDM mice were functionally defective. Our result suggests that SHARPIN plays important regulating roles in bone metabolism. These functional roles may either come from systemic chronic inflammatory or directly signaling pathway within bone cells.