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The association of SOX transcription factors expression level and breast cancer subtypes IN Esserman dataset

Supplementary Figure Legends 1-16 and Supplementary Table Legends 1-4

H3k27AC/Me3 binding peaks in the promoter area of SOX9 targets

SOX9 regulates cell apoptosis in TNBC cells but not in non-TNBC cells

Supplementary Table 1. Primers and probes for qRT-PCR assay. Supplementary Table 2. Primers for CHIP assay (positive binding sequence). Supplementary Table 3. Primers for CHIP assay (negative binding sequence). Supplementary Table 4. Primers for H3K27Ac/Me3 CHIP assay (binding peak and negative binding sequence).

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial–mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes.Implications:Our studies identified the SOX9 protein as a “master regulator” of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.

Expression level of SOXs in breast cancer cell lines

SOX9 controls G0/G1 to S phase cell cycle

SOX9-knockout suppresses TNBC cells xenograft growth in vivo

Expression of SOX9 in Curtis breast cancer dataset and NEVE breast cancer cell lines

The association of SOX transcription factors expression level and breast cancer subtypes

SOX9 expression level regulates cell migration and invasion in breast TNBC and Non-TNBC cells

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by lack of ER, PR, and Her2. Up to 85% of TNBCs have a p53 mutation as their oncogenic driver. TP53 is a tumor suppressor known as the “guardian of the genome” for its roles in regulating growth and death after genomic insult. Due to this high frequency of p53 mutations in TNBCs, targeting p53 mutants in a clinical setting is highly attractive. However, there are currently no FDA-approved drugs that can directly target p53 mutant TNBCs. We propose pathways exist that, when inhibited, will induce the specific death of p53-mutant breast cancer cells, but not p53-wild type breast cells. To investigate this hypothesis, we performed high-throughput drug screening to identify drugs that induce death in p53-mutant TNBCs. We then characterized the identified drugs for mechanism of death induction and in vivo drug effect. Methods: In vitro and in silico drug screens were conducted with small-molecule libraries of drugs with known protein targets, and screens were integrated and common drugs identified. P53-wild type and mutant cells were treated with identified drugs and stained with DAPI and DRAQ7 to identify growth-suppressive and death-inductive effects. One of these drugs, the GPX4 inhibitor ML-162, was selected for further study. Mechanism of death induction by ML-162 was determined with AnnexinV/PI, caspase cleavage, and ferroptosis assays. To confirm these results, GPX4 was knocked out and replicated the effect of ML-162. To determine the reliance of this phenotype on p53 mutational status, a series of inducible p53-mutant cell lines were created in ER+ and TNBC cell lines and then treated with small molecule inhibitors. ML-162 in vivo effect was demonstrated by treating p53-mutant TNBC xenografts in nude mice. Results: Integration of in vitro and in silico screens identified 6 common drugs, representing cell cycle inhibitors, cell division inhibitors, a proteasome inhibitor, and a peroxidase inhibitor. Identified drugs were demonstrated to reduce growth or induce death of p53-mutant TNBC cell lines. The GPX4 inhibitor ML-162 was further characterized, and found to induce death through ferroptosis, and not apoptosis or necroptosis. GPX4 knockout in p53-mutant TNBC cell lines induced ferroptosis and can be blocked with an anti-ferroptosis drug. Inducible p53-mutations in ER+ and TNBC cell lines were treated with ML-162. P53 mutations in TNBC, and not ER+, cell lines showed increased sensitivity to ML-162. To demonstrate in vivo effect of ML-162, TNBC cell line xenografts were grown in nude mice and treated with ML-162. This treatment significantly reduced tumor volume and also induced lipid peroxidation, a hallmark of ferroptosis. Conclusion: Our high-throughput screening demonstrated several of the identified drugs suppress growth or induce death preferentially in p53-mutant breast cancers. One of these drugs, ML-162, induces death of p53-mutant triple-negative breast cancer cells through induction of ferroptosis, both in vitro and in vivo. These studies provide the basic science foundation to further develop ferroptosis inducers for the targeted treatment of p53-mutant breast cancers. This work was funded by the John Charles Cain Endowment. Citation Format: William M Tahaney, Jing Qian, Reid Powell, Cassandra L Moyer, Yanxia Ma, Nghi Nguyen, Jamal Hill, Clifford Stephan, Abhijit Mazumdar, Peter JA Davies, Powel H Brown. Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells [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 GS1-09.

Background: 281,550 new cases of invasive breast cancer will be diagnosed in US this year. 15% of these patients will be diagnosed with triple-negative breast cancers (TNBCs), which is the most aggressive subtype of breast cancer. Non-specific chemotherapy remains the current standard of care for TNBCs, due to the lack of targeted therapy. We hypothesized that phosphatases are potential new drug targets for TNBCs and performed a whole phosphatase microarray analysis using TNBCs vs. estrogen receptor (ER)-positive breast cancers. NUDT5, the enzyme hydrolyzes 8-oxo-dGDP and ADP-ribose, is among the most highly expressed phosphatases in TNBCs. The goal of these studies is to investigate NUDT5 dependency and mechanism in TNBC development.Methods: In this study, we have obtained NUDT5 mRNA expression level from publicly available TCGA, METABRIC, and the CCLE datasets, and compared NUDT5 mRNA level in TNBC vs. ER-positive breast cancer. Next, we examined the association of overall survival with NUDT5 expression level dichotomized by median mRNA expression in breast cancer patients. We then engineered doxycycline-inducible NUDT5 knockdown and knockout systems in TNBC cell lines, and characterized cell growth features over 7 days with or without NUDT5 depletion. Growth inhibition effect is also confirmed by NUDT5 inhibitor. After 72 hours of doxycycline induction, cell growth was determined by cell count over 7 days. Using these TNBC cellular models, we also determined molecular phenotypes after NUDT5 depletion by immunofluorescent microscopy for 8-oxo-dGDP and γH2AX. ImageXpress Automated Cell Imaging System and CellReporterXpress were applied for image processing and data quantification. Results: NUDT5 RNA is highly overexpressed in TNBCs as compared to ER-positive breast cancers and normal breast tissue. Survival analyses indicated that increasing NUDT5 level is significantly correlated with worse clinical outcomes of breast cancer patients. NUDT5 protein is highly expressed in TNBC cell lines, compared to ER-positive breast cancer cell lines. NUDT5 knockdown or knockout both significantly inhibits the growth of TNBC cell lines. Loss of NUDT5 induced the oxidative DNA lesion 8-oxoG level in the nucleus, as well as the ratio of γH2AX-positive cells. The inhibitor of NUDT5 also suppressed TNBC cell growth in vitro. Conclusions: Our results showed that the level of NUDT5 negatively correlates with TNBC patient survival. NUDT5 is required for the TNBC cells growth and tolerance to endogenous DNA damage agents. Our findings have revealed the essential function of NUDT5, and will provide critical insights for the future development of NUDT5 inhibitors for the treatment of TNBC patients. This work was supported by Charles Cain Endowment (PB). Citation Format: Jing Qian, William Tahaney, Yanxia Ma, Abhijit Mazumdar, Powel Brown. The role of a novel phosphatase NUDT5 in triple-negative breast cancer [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 P5-12-08.

Background: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC) which lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors, that can be treated with hormone therapy, or those with HER2-positive tumors, that can be treated with anti-HER2 therapy. Thus, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated whether a novel phosphatase, NUDT5, is a potential therapeutic target. Methods: TCGA and METABRIC (Curtis) datasets were used to investigate the mRNA expression levels of NUDT5 in breast cancers. NUDT5 ablation was achieved by targeting NUDT5 with siRNA, shRNA, and sgRNA and by inhibiting NUDT5 with the small molecule inhibitor TH5427. Xenograft animal models were used to determine the effect of NUDT5 inhibition on TNBC in vivo growth. Proliferation, death, and DNA replication assays were used to investigate the cell biologic effect of NUDT5 loss or inhibition. The accumulation of 8-oxoG and the induction of gH2AX after NUDT5 loss was determined by immunofluorescence staining. Results: In this study, we demonstrated the important role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. We found that NUDT5 loss led to suppressed growth of TNBC in vitroand in vivo. This growth inhibition was not induced by death, but instead by suppressed proliferation. Loss or inhibition of NUDT5 induced an increase in 8-oxoG and gH2AX lesions in DNA and a stall in DNA replication, thus inhibiting proliferation. Conclusions: NUDT5 plays a critical role in preventing oxidative DNA damage in TNBC cells. Loss or inhibition of NUDT5 suppressed the growth of TNBCs. These biological and mechanistic studies provide a basic research foundation for the future development of NUDT5 inhibitors for the treatment of TNBC patients.

Spinal cord injury (SCI) is a devastating injury that causes permanent loss of sensation and motor function. SCI repair is a significant challenge due to the limited regenerating ability of adult neurons and complex inflammatory microenvironment. After SCI, the oxidative stress induced by excessive reactive oxygen species (ROS) often leads to prolonged neuroinflammation that results in sustained damage to the spinal cord tissue. Polydopamine (PDA) shows remarkable capability in scavenging ROS to treat numerous inflammatory diseases. In this study, we developed glial cell-derived neurotrophic factor (GDNF)-loaded PDA nanoparticle-based anisotropic scaffolds for spinal cord repair. We found that mesoporous PDA nanoparticles (mPDA NPs) in the scaffolds efficiently scavenge ROS and promote microglia M2 polarization, thereby inhibiting inflammatory response at injury site and providing a favorable microenvironment for nerve cell survival. Furthermore, the GDNF encapsulated in mPDA NPs promoted corticospinal tract motor axon regeneration and its locomotor functional recovery. Together, findings from our study revealed that the GDNF-loaded PDA/Gelatin scaffolds hold potential as an effective artificial transplantation material for SCI treatment. This article is protected by copyright. All rights reserved.

The rational fabrication of heterostructures is one of efficient strategies for improving photocatalytic performance of semiconductor photocatalysts. Very recently, Domen and co-workers found that Ta3N5 single crystals grown on the surface of KTaO3 can accomplish photocatalytic overall water splitting for the first time. In order to comprehend the underlying mechanism of this photocatalytic system, we have performed a systematic study based on density functional theory first-principles calculations. Ta3N5(010)/KTaO3(110) slab models have been built according to experimental observations by considering two common terminations of KTaO3(110) surface, named as Ta3N5/O2 and Ta3N5/KTaO. The formations of interfacial bonds are thermodynamically stable, showing a covalent interaction between two components of a heterostructure. Ta3N5/O2 has a higher mobility of photogenerated charge carriers and lower recombination rate of charge carriers than Ta3N5/KTaO. The light absorption of heterostructures displays the feature of KTaO3 in the short wavelength region and the characteristic of Ta3N5 in the long wavelength region. The calculated band offsets show that Ta3N5/O2 and Ta3N5/KTaO have distinct Type-II band alignments, with Ta3N5 as the accumulator of photoinduced electrons in the former and the collector of photogenerated holes in the latter, respectively. The difference in charge density and electrostatic potential between two components acts as a driving force to promote the transfer of electrons and holes to different domains of the interface, which is beneficial to extend the lifetime of photoinduced carriers. Our results demonstrate that the function of Ta3N5 in Ta3N5/KTaO3 photocatalytic system is determined by the termination property of KTaO3(110) surface, which provides a likely reason of the observed photocatalytic activity of overall water splitting achieved by Ta3N5 synthesized by using KTaO3 as a precursor for the nitridation reaction.

Tetracycline is extensively used as an antibiotic in animal husbandry, and there arose an increase in antibiotic resistance genes in the environment, posing a threat to human health. Motivated by this, a magnetic molecularly imprinted material based on synergistic recognition (1 + 12) was constructed and coupled with high-performance liquid chromatography to detect ultra-trace tetracycline in complicated samples. In this case, the molecularly imprinted polymers were synthesized via a "one-pot" method and acted as recognition elements on the surface of silica-coated ferroferric oxide particles. Aptamers and β-cyclodextrin, as functional monomers, had a synergistic effect on the recognition of tetracycline and the synergistic recognition factor was 1.7. Meanwhile, the material exhibited high selectivity to tetracycline with an imprinting factor of 7.6. In addition, compared to being modified on the surface, the stability of the aptamers was effectively improved by cross-linking in the molecularly imprinted polymer framework. Relevant experimental conditions, such as buffers, concentration of magnesium ions and adsorption time, were optimized. As a result, the method showed a limit of detection of 1.0 μg L

Codoping with cations and anions is found to be an effective approach to tailor the electronic structures of semiconductor-based photocatalysts. In this work, a systematic hybrid density functional study has been carried out for BaTiO3 with a wide bandgap, codoped with TM and X (TM = V, Nb, Ta, Mo and W, X = N and C), with the objective of improving its photocatalytic activity for water splitting under visible light irradiation. Oxygen-rich conditions are found to be more favorable for the synthesis of the (TM + X)-codoped materials, and under these conditions, the formation energy for the codoped BaTiO3 is even lower than that of the TM-monodoped system. A smaller deformation of the BaTiO3 crystal structure corresponds to a more favorable formation of (TM + X)-codoped systems. Passivated codoping is found to be advantageous because it effectively reduces the band gap without encountering any localized impurity states observed in the related monodoped systems. The band alignments for all the codoped systems are well positioned for the feasibility of both photooxidation and photoreduction of water. It is found that there are seven investigated systems with bandgaps smaller than 3 eV. Among them, passivated codoped systems including Mo and W have been found to be the most effective for narrowing the bandgap (Eg is in the range of 1.55–2.16 eV), increasing the mobilities of photoinduced carriers and improving the photocatalytic activity of BaTiO3 under visible light. These materials are promising photocatalysts for overall water splitting under visible-light irradiation and further experimental investigations are highly demanded to explore their potential applications in the photocatalytic field.

Purpose Xiaoai Jiedu recipe (XJR), a formula long used by Chinese National Medical Professor Zhou Zhongying, has potent antitumor properties, but the molecular mechanism is still unclear. The aim of the study was to investigate the antitumor mechanism of XJR on hepatocellular carcinoma (HCC) by focusing on miRNA. Methods Three concentrations of XJR (low, middle, and high) were used to treat tumor xenograft mice models. Microarray technology was used to identify the differential expressed genes after XJR treatment, and bioinformatic tools and luciferase reporter assay to predict the potential pathways. HepG2 cells were transfected with inhibitor of miR-200b-3p to detect the effect of miR-200b-3p and Notch1 on tumor growth. Results XJR effectively exerted anti-HCC effect both in vitro and in vivo. MiRNA chip analysis results showed that the expression of 75 miRNAs was upregulated and 158 miRNAs was downregulated in blood from XJR-treated mice. Further validation by using real-time polymerase chain reaction (RT-PCR) assay showed that the expression of five miRNAs (miR-453, miR-200b-3p, miR-135a-1-3p, miR-1960, miR-378a-5p, and miR-466f) was consistent with the results of miRNA chip analysis. Among them, miR-200b-3p was selected as candidate for further research. Results of the MTT, migration, and wound healing assays showed that down-expression of miR-200b-3p abrogated the effect of XJR on cell growth and metastasis. Luciferase reporter assay confirmed that Notch1 was the direct target of miR-200b-3p. XJR significantly decreased Notch1 expression in HepG2 cells, whereas miR-200B-3p inhibitor abrogated the XJR-induced decrease in Notch1 expression. Conclusion This study indicated that XJR could effectively inhibit HCC and might exert its antitumor effect through the miR-200b-3p/Notch1 axis. These findings provided new avenues for the use of XJR for prevention and treatment of HCC.

Glioma is the most common and deadly tumor in central nervous system. According to previous studies, long noncoding RNAs (lncRNA) and transcription factors were significant factors of gliomas progression by regulating gliomas immune microenvironment. In our study, we built two independent cohorts from CGGA and TCGA. And we extracted 253 immune-related lncRNA correlated with prognosis. After LASSO analysis and multivariate Cox regression analysis, 8 immune-related lncRNA were used to construct classifier. The effectiveness of classifier was confirmed in both CGGA (AUC = 0.869) and TCGA (AUC = 0.902) cohorts. The correlation between transcription factors and immune-related lncRNA was calculated by WCGNA. Eventually, we built a network between 8 lncRNA and transcription factors. The function of core immune-related lncRNA in gliomas immune microenvironment was also investigated by CIBERTSORT. Our research provided a strong classifier of immune-related lncRNA to predict gliomas patient outcome. We also found the correlation between core immune-related lncRNA and transcription factors. These results may stimulate new strategy of immunotherapy in gliomas patients.

The construction of effective and safe theranostics was very important to realize tumor diagnosis and treatment simultaneously. Photothermal therapy had obtained great attention due to its remarkable capability of both cancer treatment and biosecurity. Molybdenum disulfide (MoS2) nanoflakes have shown excellent photothermal conversion efficiency, which were explored to photothermal therapy. However, MoS2 nanoflakes would decrease or quench the intensity of fluorescence when the MoS2 nanoflakes directly incorporated with fluorescence dyes to construct bioimaging theranostic systems. In this work, MoS2 nanoflakes embedded into MSNs to construct photothermal nanomaterials, which realized the nanoparticle size controllable. Fluorescein isothiocyanate (FITC) conjugated with mesoporous silica nanoparticles via amide bonds for bioimaging. It was more stable than physical absorption. As expected, the as-prepared FITC–MoS2 MSNs possessed photothermal effect, fluorescence stability and photostability. FITC–MoS2 MSNs were quickly taken up by HepG2 cells. Meanwhile, the HepG2 cells could be efficiently ablated under the 808 nm near-infrared laser irradiation. These results proved that we successfully constructed multifunctional MSNs for tumor theranostics. It also provided a potentially common nanoplatform for theranostic of biomedical applications.

Unique optical, electrical and chemical properties make carbon nanotubes (CNTs) an excellent candidate for potential applications in the next-generation optoelectronics. Especially, the optoelectronic properties of CNTs can be enhanced dramatically by constructing heterostructures with other materials, in which the charge separation efficiency is enhanced and the recombination probability of excitons is suppressed significantly. Therefore, the CNT-based heterostructures have been widely used as active materials in high-performance photoelectronic devices. Herein, the recent progress of the CNT-based heterostructure photodetectors is reviewed. Firstly, the working mechanisms and typical figures-of-merits are introduced. Secondly, different type CNT-based heterostructures and related photodetectors are highlighted, such as van der Waals heterostructures, all-carbon heterostructures, and bulk heterostructures. Finally, we give the current challenges and future prospects for the development of this emerging field.

Colon cancer stem cells (cCSCs) are highly tumorigenic and resistant to traditional chemotherapeutic drugs. Therefore, they are an essential factor in colorectal cancer (CRC) metastasis and recurrence. Dendritic cells (DCs) could bind to the tumor cells and form the fusion cells (FCs). And these FCs could inhibit the development of malignant tumors. Furthermore, the cytokine induced killer (CIK) cells (CD3+ /CD56+ T lymphocytes) could also apply for the immunotherapy of cancer. And compound Kushen injection (CKI) is a traditional Chinese medicine (TCM) which has been used for the treatment of various tumors. However, whether the dendritic-colon cancer stem cell fusion cells (DC-cCSC FCs) could activate the CIK cells and kill the colon cancer stem cells is unknown. And whether the CKI could enhance the lethal effect is still unclear. In this study, we collected peripheral blood samples from healthy participants to acquire mononuclear cells and induced DC and CIK cells. Meanwhile, the CD44+ cells (cCSCs) were screened from SW480 cells. Next, the DC-cCSC FCs were established for the next experiments. At last, CCK-8 assays were performed to determine the effect of DC-cCSC FCs and CKI on the viability of cCSCs. We found that DC-cCSC FCs enhanced the proliferation of CIK cells and induce the CIK cells to secrete more IL-12. The DC-cCSC FCs enhanced the inhibitory effect of CIK cells on cCSCs. Furthermore, application of CKI enhanced the killing rates of DC-cCSC FCs and CIK cells on cCSCs. CIK cells activated by the DC-cCSC FCs had the lethal effect on the cCSCs. Furthermore, CKI enhanced this lethal effect of DC-cCSC FCs and CIK cells.

β-Ge3N4 loaded with nanoparticulate RuO2 as a cocatalyst is the first successful non-oxide photocatalyst for overall water splitting. To get an insight into the working mechanism of this particular photocatalytic system, we have calculated geometrical structures of low-index surfaces for β-Ge3N4. Analysis of surface energies indicates that the most preferentially exposed surface is (100). The band gap of surface is narrower than that of bulk due to the dangling bonds. Dissociative water adsorption on (100) surface is thermodynamically favorable. The adsorption behavior of (RuO2)n (n = 2, 3, and 4) clusters on the β-Ge3N4(100) surface has been explored. It is found that all the clusters bind to (100) surface strongly by forming interfacial bonds so that the adsorptions are exothermic processes. The calculation on density of states for β-Ge3N4(100) surface loaded with (RuO2)n clusters reveals that photo-induced electrons tend to accumulate on (RuO2)n clusters and holes tend to stay in β-Ge3N4. Based on the theoretical indication of Type-II staggered band alignment, we have proposed that in photocatalytic water splitting reaction, oxygen evolution reaction is inclined to occur on the surface of β-Ge3N4 while hydrogen evolution reaction is apt to occur on (RuO2)n clusters. In a word, loading RuO2 nanoparticles as a reduction cocatalyst benefits the charge separation in β-Ge3N4. Furthermore, attaching (RuO2)n clusters onto β-Ge3N4(100) surface results in the redshift of absorption edge and the increase of absorption intensity. Our calculations have reasonably explained the experimental observation on the decomposition of water into H2 and O2 after loading RuO2 cocatalyst in β-Ge3N4 photocatalyst.

Because of the limited regenerative ability of the central nervous system (CNS), effective treatments for spinal cord injury (SCI) are still lacking. After SCI, neuron loss and axon regeneration failure often result in irreversible functional impairment. The calcium overload induced by the N-methyl-D-aspartate receptor (NMDAR) overactivation is critical for cell death in SCI. It has been reported that the magnesium ion (Mg

Background Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, characterized high rates of tumor protein 53 (p53) mutation and limited targeted therapies. Despite being clinically advantageous, direct targeting of mutant p53 has been largely ineffective. Therefore, we hypothesized that there exist pathways upon which p53-mutant TNBC cells rely upon for survival. Methods In vitro and in silico drug screens were used to identify drugs that induced preferential death in p53-mutant breast cancer cells. The effects of the glutathione peroxidase 4 (GPX4) inhibitor ML-162 was deleniated using growth and death assays, both in vitro and in vivo. The mechanism of ML-162 induced death was determined using small molecule inhibition and genetic knockout. Results High-throughput drug screening demonstrated that p53-mutant TNBCs are highly sensitive to peroxidase, cell cycle, cell division, and proteasome inhibitors. We further characterized the effect of the Glutathione Peroxidase 4 (GPX4) inhibitor ML-162 and demonstrated that ML-162 induces preferential ferroptosis in p53-mutant, as compared to p53-wild type, TNBC cell lines. Treatment of p53-mutant xenografts with ML-162 suppressed tumor growth and increased lipid peroxidation in vivo. Testing multiple ferroptosis inducers demonstrated p53-missense mutant, and not p53-null or wild type cells, were more sensitive to ferroptosis, and that expression of mutant TP53 genes in p53-null cells sensitized cells to ML-162 treatment. Finally, we demonstrated that p53-mutation correlates with ALOX15 expression, which rescues ML-162 induced ferroptosis. Conclusions This study demonstrates that p53-mutant TNBC cells have critical, unique survival pathways that can be effectively targeted. Our results illustrate the intrinsic vulnerability of p53-mutant TNBCs to ferroptosis, and highlight GPX4 as a promising target for the precision treatment of p53-mutant triple-negative breast cancer.

Background: Triple-negative breast cancers (TNBCs) are the most aggressive breast cancers and have a very poor prognosis. Since TNBCs lack the expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), development of new TNBC treatment strategies is an urgent clinical need. Our previous studies have demonstrated that high expression and activity of SOX9 transcription factor positively related to TNBC cell growth and tumor metastasis in vivo. It may be possible to target the SOX9 transcription factor by targeting upstream SOX9-activating proteins. Hypothesis: SOX9-binding protein, S100A10, regulates SOX9 activity and control TNBC growth. Material and Methods: Immunoprecipitation (IP) in combination with mass spectrometry (IP-MS) analysis was used to identify SOX9 binding proteins, with support from MD Anderson’s proteomics core. The Mascot Score is a statistical score used as a measure of the reliability of identifying a specific protein by IP-MS. The data was summarized based on the Mascot Score. IP-Western Blotting analysis was used to confirm that S100A10 bound SOX9 in TNBC cells. We then treated TNBC cells with or without siRNAs of S100A10 to evaluate its effect on TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. SOX9 activity on regulating β-catenin pathway was measured using TOP/FOP luciferase activity analysis. Data are presented as mean values ± SD. Statistical significance was calculated using the Student’s t-test unless otherwise indicated. Results: Using immunoprecipitation combined with mass spectrometry (IP-MS), we identified multiple SOX9-binding proteins. One of the identified SOX9-binding proteins was S100A10 (S100A10). Knockdown of S100A10 decreased TNBC cell growth in vitro. Using TOP/FOP luciferase activity analysis, we demonstrated that S100A10 regulates the β-catenin pathway. Thus, S100A10 is a potential activator of SOX9 that can be targeted to inhibit SOX9 downstream signaling and TNBC growth. Conclusion: Our results demonstrate that S100A10 is a SOX9-binding protein, that regulates SOX9 downstream signaling and controls TNBC cell growth. Implications: Inhibition of S100A10 represents a novel strategy to treat TNBCs. S100A10 inhibitors should be developed as promising new drugs to treat these aggressive breast cancers. Grant Support: These studies were supported by a Breast Cancer Research Foundation (BCRF) grant (PB). The Proteomics and Metabolomics Facility was supported in part by Cancer Prevention Research Institute of Texas (CPRIT) grant number RP130397 and NIH grant numbers 1S10OD012304-01 and P30CA016672. Citation Format: Yanxia Ma, Jing Qian, Cassandra Moyer, Amanda Lanier, Jamal Hill, Darian Coleman, David H. Hawke, Abhijit Mazumdar, Powel H. Brown. Targeting S100A10, a SOX9 binding protein, for the treatment of triple negative breast cancer. [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 3966.

Salvianolic acid A (Sal A) has a wide range of pharmacological activities. To date, there have been no systematic and detailed metabolite research data of Sal A after oral administration in vitro and in vivo. In this study, a rapid and systematic method based on ultrafast liquid chromatography-quadrupole-time-of-flight mass spectrometry was developed to detect metabolites of Sal A in vitro (human liver microsome, human intestinal microbiota, artificial gastric, and intestinal juice) and in vivo (urine, plasma, feces, and various organs collected after oral administration of Sal A to normal rats and pseudo-germ-free rats). A total of 26 metabolites of Sal A were characterized. These metabolites were formed through extensive metabolic reactions, such as hydroxylation, hydrogenation, and glucuronidation reactions. This study provides novel possibility for exploring the potential biological mechanism of Sal A, and aids the promotion of clinical application.

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells’ (MSCs’) biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.