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Triple negative breast cancers (TNBCs) lack estrogen and progesterone receptors and Her2/neu amplification, and are hence not treatable with therapies targeting these molecules. TNBCs have upregulated DNA damage response mechanisms, including the Rad6 postreplication repair (PRR) pathway, that potentially contribute to chemoresistance. Rad6 is a major component of the PRR pathway and its ubiquitin conjugating (UBC) activity is critical for its function. Rad6 expression is low in normal breast cells and tissues but the Rad6 homolog Rad6B is overexpressed in invasive, metastatic and chemoresistant BrCas. Constitutive overexpression of Rad6B in MCF10A cells induces resistance to cisplatin and doxorubicin. TCGA analysis of TNBC patient data showed an association between high Rad6B expression (but not Rad6A) and decreased overall survival. We recently reported the development of a novel Rad6-selective small molecule inhibitor (SMI#9) that inhibits Rad6 UBC activity, migration, and induces apoptosis in TNBC cells but has no effect on MCF10A cells. Since SMI#9 has limited aqueous solubility, in this study we synthesized a modified analog of SMI#9 to enable conjugation via a hydrolyzable ester bond to gold nanoparticle (GNP) and to improve delivery. GNP tethered SMI#9 (SMI#9-GNP) was characterized for purity, ligand conjugation and size by thermogravimetric analysis, atomic force microscopy, transmission electron microscopy, UV-Vis spectroscopy and zeta sizer, and for cellular uptake and drug release by FTIR and mass spectrometry. We compared the activities of SMI#9-GNP and free SMI#9 for cytotoxicity and intracellular localization in mesenchymal (MDA-MB-231 and SUM1315) and basal (MDA-MB-468 and HCC1937) subtypes of TNBC, and in MCF10A cells. Whereas free SMI#9 was cytotoxic to all TNBC cells, SMI#9-GNP demonstrated as good or better cytotoxicity than free SMI#9 only in mesenchymal TNBC cells. MCF10A cells were unaffected by both free and SMI#9-GNP. Consistent with cellular sensitivities, SMI#9-GNP is efficiently endocytosed and processed in lysosomes in mesenchymal TNBC cells, while uptake into basal TNBC cells is compromised by cell microenvironment induced SMI#9-GNP aggregation. SMI#9-GNP treatment induces mitochondrial dysfunction, and stabilization and hyperactivation of PARP-1 that was commensurate with autophagy (indicated by LC3-I to LC3-II conversion). Rad6 loss and PARP-1 hyperactivation are associated with mitochondrial dysfunction, and since inhibition of Rad6 induces both mitochondrial dysfunction and PARP-1 activation this implicates a potential novel role for Rad6 in linking these processes. In summary, our data show that SMI#9-GNP is a suitable delivery vehicle and that the SMI#9 released from GNP conjugate functions similarly as free SMI#9. Our data also illustrate how cell microenvironment induced changes in the physical properties of GNP-drug conjugates can have important implications in the application of nanoparticles in cancer therapy. Supported by NIH R21 CA178117. Citation Format: Haynes B, Zhang Y, Li J, Petit S, Westwell A, Mao G, Shekhar M. Evaluation of the therapeutic efficacy of a Rad6 small molecule inhibitor in triple negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-06.

Cell surface molecules expressed on cancer cells can be used as diagnostic and therapeutic tools. In breast cancer, CD44 and CD24 were identified as cell surface markers characterizing cancer cell stemness that may correlate with prognosis. Breast cancer cells with high CD44 and low CD24 expression have been shown to exhibit proliferative, invasive and metastatic properties that may relate to drug sensitivity and metastatic risk in patients. In addition to stemness, immune evasion is another hallmark of cancer with CD47 “don’t eat me” and CD274 “don’t find me” molecules playing important roles in antitumor immunity. Although each of these molecules are of interest as therapeutic targets, current therapeutic paradigms involve multimodal approach with radiation therapy often having a central role. Hence, the effect of irradiation, and related radio-sensitizers, on the expression of cancer cells surface markers to be targeted with an adjuvant therapeutic mode must be considered when designing such therapies. The goal of this study was to determine the dynamics of CD44, CD24, CD47 and CD274 expression in radio-sensitized and irradiated MDA-MB 231 breast cancer cells. We have recently reported that gold nanoparticles (AuNPs) sensitized breast cancer cells to irradiation at kV and MV energies. Here, we hypothesize that AuNP may also modulate the irradiation altered expression of cell surface markers. We explored this by using 10 MV energies and 6 Gy radiation dose. MDA-MB-231 cells were incubated for 3 hours with 14nm AuNPs, irradiated and allowed to grow for 24h and 72h after which percenteges of positive cells were determined by flow cytometry. Results are expressed as a percent of control, non-irradiated cells that was set at 100%. At 24h non-irradiated cells pre-incubated with AuNP exhibited a decrease in the percentage of CD24+ and CD44+ cells, while no significant change in the percentage of CD47+ and CD274+ cells was observed. Irradiation with 6Gy at 10 MV induced a decrease in CD24+ and an increase in CD44+ cells and AuNP potentiated this effect. However, 72h post-irradiation, percentages of CD24+ and CD44+ cells significantly increased, compared to non-irradiated controls and this effect was of lesser magnitude in AuNP pretreated cells. Percentage of CD47+ and CD274+ cells also significantly increased 24h post-irradiation, but with less magnitude in AuNP treated cells. At 72h in AuNP treated irradiated cells no change was observed in the percentage of CD274+ cells, while the percentage of CD47+ cells significantly increased in comparison to the irradiated cells not exposed to AuNPs. Preliminary data shown here indicate that expression of molecules important for cancer progression, metastasis and immune evasion undergo changes in response to irradiation, and that these changes are affected by AuNPs. Further studies will shed more light on the mechanisms behind these observed effects. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin Chetty, Benjamin Movsas, Ning Wen. Effect of irradiation and gold nanoparticle on expression dynamics of cell surface markers in MDA-MB 231 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4862.

Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin J. Chetty, Benjamin Movsas, Ning Winston Wen. Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1376.

Active β-catenin signaling in melanoma is associated with aggressive disease. We have previously shown that Rad6B, an ubiquitin conjugating enzyme, is a positive regulator of β-catenin stability and transcriptional activity, and is itself a transcriptional target of β-catenin. This stabilization is mediated by Rad6B-induced K63-linked polyubiquitination of β-catenin that renders it insensitive to 26S proteasomal degradation. Immunohistochemical analysis of human cutaneous melanoma samples showed the first detectable expression of Rad6B in melanocyte hyperplasia and continued Rad6B and β-catenin overexpression in primary and metastatic melanomas. These data implicate a critical role for Rad6B in melanocyte transformation and a continuing role in melanoma progression. To analyze the role of Rad6B in melanomagenesis, we targeted Rad6B expression/activity in human M14 metastatic melanoma cells by using a Rad6-selective small molecule inhibitor SMI#9 and the CrispR/Cas9 gene editing system. Immunoblot analysis showed that compared to controls, M14 cells treated with SMI#9 showed decreased levels of β-catenin, and β-catenin driven transcriptional targets Rad6, vimentin, Mitf-M, and Melan A. Although Snail levels were not altered, SMI#9 treated cells showed increase in phosphorylated Snail. Immunocytochemical staining verified the western blot data and showed cytoplasmic relocalization of PCNA and Snail in SMI#9 treated cells which is consistent with increase in phosphorylated Snail. Concordant with the decrease in vimentin and unphosphorylated Snail levels, Rad6B-inhibited cells display poor capacity to migrate as compared to controls. Whereas Rad6B knockout cells were severely growth impaired, Rad6B edited clones showing partial expression (potentially from only one allele) similarly showed decreased levels of β-catenin, vimentin, and Mitf-M. Similar to Rad6B-inhibited cells, Rad6B edited cells were migration- and invasion-impaired. Whereas control cells produced robust tumors and overt lung metastases, the Rad6B gene edited cells were poorly tumorigenic. Rad6B gene edited clones showed complete loss of Melan A. Since Mitf-M is a transcriptional regulator of Melan A and a key player in melanocyte lineage specification, our data suggest that targeting Rad6B could potentially inhibit melanomagenesis by reprogramming melanoma cells. Supported by NIH CA178117 and 3Balls Racing. Citation Format: Ashapurna Sarma, Sarah Petit, Fangchao Liu, Pratima Nangia-Makker, Guangzhao Mao, Malathy Shekhar. Targeting Rad6B suppresses melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1149. doi:10.1158/1538-7445.AM2017-1149

Maspin, an epithelial-specific member of the serine protease inhibitor superfamily, inhibits tumor invasion and metastasis. Overall maspin expression is downregulated or lost in invasive and metastatic carcinomas. Maspin subcellular distribution among the nuclear, cytoplasmic and extracellular compartments is differentially regulated during tumor progression. Extracellular maspin has been shown to inhibit tumor cell invasion and motility in vitro. Also, recombinant maspin was shown to inhibit endothelial cell tube formation in vitro and tumor-angiogenesis in vivo. To date, the mechanisms governing maspin trafficking remain unclear. In the current study, we aim to investigate the mechanism and biological significance of maspin secretion. Normal and tumor cells known to secrete maspin, were treated with or without drugs targeting classical or non-classical secretory pathways. The CM were collected and analyzed for soluble and exosome-associated proteins. We also utilized atomic force microscopy to characterize the physical characteristics of exosomes. We found maspin as a soluble protein in the CM. Maspin secretion was not inhibited by the drug treatments. In fact, maspin secretion increased under drug treatment targeting the classical secretory pathway. Furthermore, maspin was detected in the exosomes. Under drug treatment, the overall increase in maspin secretion was accompanied by an increase in detection of maspin in the exosomes. Based on the drug treatment-induced changes in maspin's secretion patterns, we speculate that maspin is secreted via a non-classical pathway. In addition, we provide the first evidence that maspin is secreted inclusively as both a free and an exosome-encapsulated molecule in normal and tumor cells. The existence of extracellular maspin as a free and an exosome-associated molecule implies a dual role for maspin in the tumor microenvironment. These novel findings are currently under investigation since the tumor suppressive activity of exosome-associated maspin may be of particular significance in cell-cell communication, and tumor suppressive activity of free maspin may be associated with extracellular matrix remodeling. Citation Format: Ivory S. Dean, Sijana Dzinic, Alexander Kaplun, Yi Zou, Guangzhao Mao, Shijie Sheng. Human prostate epithelial cells secrete the tumor suppressor maspin as a soluble and exosome-associated protein . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2013-5067