Combination effects of Pistachio hull and carfilzomib on NF-κB p65, MDR1, MRP1, and Caspase3 gene expression in breast cancer cell line

Abstract #1039 Introduction: The HER family of receptors HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) is critical in the regulation of cell growth, proliferation and survival. Several studies have linked over-expression of HER1 and HER2 with resistance to chemotherapy and radiation. Clinical trials combining the HER tyrosine kinase inhibitors (TKI) gefitinib (GEF), an inhibitor of HER1, and lapatinib (LAP), a dual inhibitor of HER1 and HER2, with chemotherapeutic agents have not shown synergy comparable to that found in preclinical models. We investigated the effects of chronic treatment of breast cancer cells with TKI inhibitors on DNA damage and repair resulting from chemotherapy and ionising radiation (IR).
 Material and Methods: Western blotting was used to examine the expression of phosphorylated HER receptors, AKT and MAPK in the SKBR3 and BT474 human breast cancer cell lines. Etoposide (ET), doxorubicin (DOX) and IR induced DNA strand breaks (SB) were measured in the SKBR3 cell line using the single cell gel electrophoresis (comet) assay. Cisplatin-induced (PT) interstrand crosslinks (ICL) were measured with the modified alkaline comet assay. Cells were pre-treated with LAP or GEF for 1 hour or 48 hours, and TKI was replaced at 24-hourly intervals. Cell cycle analysis and intracellular DOX were measured by flow cytometry.
 Results: In the SKBR3 and BT474 cell lines pHER1, pHER2, pHER3, pAKT and pMAPK were inhibited within 1 hour of exposure to LAP or GEF. However, after 48 hours, pHER3 and pAKT signalling was detected in cells treated with GEF, but not LAP. pHER1, pHER2 and pMAPK were inhibited for at least 72 hours with both drugs. Cell cycle analysis on the SKBR3 cell line showed that 48 hours exposure to either TKI reduced the number of cells in S (12.4±1.1% to 3.2±0.3%), and G2 (16.0±0.55% to 7.1±1.0%) phases of the cell cycle. There was no alteration in intracellular DOX levels at 1 hour or following 48 hours exposure to either TKI. SB were induced by ET, DOX and IR, and ICL by PT, in cells exposed to GEF or LAP for 1 hour. Exposure for 48 hours did not alter the number of ICL or SB induced by PT or IR. In contrast there was a 30-90% reduction in SB following ET treatment (depending on drug concentration) in cells treated with TKI for 48 hours compared with 1 hour treatment. The effect of 48 hours exposure to TKI on DOX-induced SB was more marked with >90% reduction, even at high DOX concentrations. Higher concentrations of ET resulted in increased SB, but increasing concentration of DOX did not alter the level of detectable SB.
 Discussion: These results suggest why clinical trials of combinations of HER TKI with chemotherapy agents fail to show the magnitude of synergy predicted in preclinical studies. Whilst the cytostatic properties of both LAP and GEF may explain the reduction in DNA damage after 48 hours, this does explain why SB are absent in DOX-treated cells. Further studies investigating kinetics of DNA repair following chemotherapy or radiation in combination with TKI are on-going. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1039.

Identifying novel molecular drug targets continues to be of prime interest in addressing the public health burden of breast cancer in both developed and developing countries alike. In this context, proteomics/pharmacoproteomics approaches offer a new dimension for personalized medicine. We have previously identified differentially expressed proteins with antigenic activity between SKBR3 (ER-, high HER2 expression) and MCF7 (ER+, low HER2 expression) breast cancer cell lines. The aims of the present study were (1) to develop an initial proteome based roadmap of differentially expressed proteins between the two cell lines using two-dimensional electrophoresis (2-DE), and (2) to compare them to those identified by other techniques. SKBR3 and MCF7 cell lysates were subjected to 2-DE and spots of interest were identified by MALDI-TOF/TOF MS. Upregulated proteins (≥2 fold and p<0.05) in MCF7 cells were cellular retinoic acid binding-protein-2, Hsp27, nucleophosmin, electron transfer flavoprotein-α, and profilin-2. In SKBR3 cells, upregulated proteins were RhoGDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel-2, aldehyde dehydrogeanase-2 (ALDH2), LDH-A, LDH-B, pyrophosphates-1, GAPDH, cathepsin-D preprotein, F–actin capping protein β-subunit, and apolipoprotein A-I binding protein. Differential expression of RhoGDI-α, a molecule with a versatile range of biological activities in different types of breast cancer, was validated using western blotting. In conclusion, these observations using proteomics strategies serve to characterize SKBR3 and MCF7 breast cancer cell lines and offer new insights for personalized medicine on differential expression of putative drug targets between these cancer models. Further studies are warranted to examine the usefulness of SKBR3 cell line as an appropriate model for studying RhoGDI-α activities in HER2+ ER- breast cancer. Finally, we underscore that the findings presented herein also attest to an emerging strand of collaborative proteomics/OMICS studies in developing countries and resource-limited settings towards global personalized medicine, an area of postgenomics data-intensive health research that is in need of greater attention in biomedical literature. Keywords: Breast cancer, developing world OMICS, ER, HER2, personalized medicine and developing countries, pharmacoproteomics, proteomics, RhoGDP dissociation inhibitor-α

Differentially expressed proteins among cancer cell lines fit each cell line as a model for gaining knowledge of heterogeneity in cancer. The aim of our study was to identify differentially expressed proteins between MCF7 (ER+, low HER2 expression) and SKBR3 (ER-, high HER2 expression) cell lines by a proteomic approach. Moreover, a number of proteins in MCF7 cell line were randomly selected and identified. MCF7 and SKBR3 cell lysates were subjected to two dimensional gel electrophoresis and spots of interest were identified by MALDI-TOF/TOF mass spectrometry. Upregulated proteins (≥2 fold and p value &amp;lt;0.05) in MCF7 cells were cellular retinoic acid binding-protein 2, Hsp27, nucleophosmin, electron transfer flavoprotein ≤ subunit, and profilin, and in SKBR3 cells were Rho GDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel 2, aldehyde dehydrogeanase-2 (ALDH2), LDH- A, LDH-B, pyrophosphates 1, GAPDH, cathepsin D preprotein, and apolipoprotein A-I binding. Most of the identified proteins have been a candidate marker for cancer aggressiveness or drug resistance, but their differential expressions between SKBr3 and MCF7 cells were not known. Apo-lipoprotein binding-protein has not been described in cancer so far. Differential expression of RhoGDI-α was further validated by using western blotting with specific antibody. Further studies are required to clarify the importance of differential expressions of the identified proteins in SKBr3 and MCF7 breast cancer cell line models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4785. doi:1538-7445.AM2012-4785

Introduction: Trastuzumab (T) is a monoclonal antibody therapy used in the treatment of HER2+ breast cancer. T inhibits HER2 intracellular signalling and is capable of engaging the immune system through ADCC. Adenosine is an important negative regulator of the immune response through its interaction with the A2A receptor (A2AR, ADORA2A). Relieving adenosine-mediated immunosuppression by inhibiting A2AR may improve NK cell-mediated T-ADCC against HER2+ breast cancer cells. In addition, we have previously shown that SKBR3 cells resistant to the EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib are less sensitive to T-ADCC and showed increased A2AR protein levels. This study examines the effects of inhibiting A2AR signalling on NK cell-mediated T-ADCC against treatment naïve HER2+ breast cancer cell lines HCC1954 and SKBR3 and lapatinib and afatinib (irreversible pan-HER-family TKI)-resistant sublines of HCC1954 and SKBR3. Methods: HER2+ breast cancer cell lines SKBR3 and HCC1954 were exposed to afatinib (150nM) or lapatinib (1μM) for 6 months to generate TKI-resistant SKBR3-A and HCC1954-L cell lines. Acid-phosphatase-based proliferation assays were used to confirm resistance to TKI treatment. Western blotting was used to examine A2AR and HER2 protein levels in cell lines. NK cells were isolated from healthy volunteer whole blood by MACSxpress isolation kits. Immune cell-mediated cytotoxicity was determined at a 1:1 (NK cell: TC) ratio over 12 hours using a flow cytometry-based method. Direct cytotoxicity and T-ADCC were determined +/- A2AR agonist CGS21680 (1 μM) and/or A2AR antagonist preladenant (100 nM) for all cell lines. Experiments were carried out three times with three separate volunteer samples with representative results presented. Results: HCC1954-L cells were 5.3-fold resistance to lapatinib (IC50 1.65 μM +/- 0.22 μM) vs. HCC1954 (IC50 0.31 μM +/- 0.15 μM). SKBR3-A cells were 33-fold resistant to afatinib (IC50 0.28 μM +/- 0.006 nM) vs. the parental SKBR3 cell line (IC50 0.009 μM +/- 0.006 μM). SKBR3 and HCC1954 expressed detectable protein levels of A2AR. A2AR and HER2 levels were not significantly changed between parental and resistant cell lines. Levels of direct cytotoxicity and T-ADCC elicited by NK cells were higher against SKBR3-A (p=0.002) and HCC1954-L cells (p=0.0004) than parental cell lines. The A2AR agonist CGS21680 alone had inconsistent effects on direct cytotoxicity and T-ADCC in all cell lines tested. The addition of A2AR antagonist preladenant to CGS21680, but not preladenant alone, increased T-ADCC against the parental HCC1954 cells by 12.7 +/- 3.4% and parental SKBR3 cells by 9.5 +/- 3.6%. T-ADCC levels in the targeted therapy-resistant HCC1954-L and SKBR3-A cell lines were not impacted by the CGS21680/preladenant combination. Conclusions: A HER2-targeted therapy resistance phenotype is associated with increased T-ADCC in the models tested. Inhibition of activated A2AR can increase T-ADCC elicited by NK cells against treatment naïve HER2+ breast cancer cell lines but not TKI-resistant sublines. Further work is warranted to examine the impact of targeting A2AR in HER2+ breast cancer. Citation Format: Gaynor N, Noone J, Monedero J, Murphy EE, O'Gorman DJ, Crown J, Collins DM. The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-19.

HER2-positive breast cancer is one of the most prevalent forms of cancer among women worldwide. Generally, the molecular characteristics of this breast cancer include activation of human epidermal growth factor receptor-2 (HER2) and hormone receptor activation. HER2-positive is associated with a higher death rate, which led to the development of a monoclonal antibody called trastuzumab, specifically targeting HER2. The success rate of HER2-positive breast cancer treatment has been increased; however, drug resistance remains a challenge. This fact motivated us to explore the underlying molecular mechanisms of trastuzumab resistance. For this purpose, a two-fold approach was taken by considering well-known breast cancer cell lines SKBR3 and BT474. In the first fold, trastuzumab treatment doses were optimized separately for both cell lines. This was done based on the proliferation rate of cells in response to a wide variety of medication dosages. Thereafter, each cell line was cultivated with a steady dosage of herceptin for several months. During this period, six time points were selected for further in vitro analysis, ranging from the untreated cell line at the beginning to a fully resistant cell line at the end of the experiment. In the second fold, nucleic acids were extracted for further high throughput-based microarray experiments of gene and microRNA expression. Such expression data were further analyzed in order to infer the molecular mechanisms involved in the underlying development of trastuzumab resistance. In the list of differentially expressed genes and miRNAs, multiple genes (e.g., BIRC5, E2F1, TFRC, and USP1) and miRNAs (e.g., hsa miR 574 3p, hsa miR 4530, and hsa miR 197 3p) responsible for trastuzumab resistance were found. Downstream analysis showed that TFRC, E2F1, and USP1 were also targeted by hsa-miR-8485. Moreover, it indicated that miR-4701-5p was highly expressed as compared to TFRC in the SKBR3 cell line. These results unveil key genes and miRNAs as molecular regulators for trastuzumab resistance.

BackgroundThe MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism.ResultsA range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines.ConclusionsOur study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.

Toxicity of trastuzumab labeled 177Lu on MCF7 and SKBr3 cell lines

Introduction Due to the unique conditions of the tumor microenvironment, such as hypoxia, poor angiogenesis, and mitochondrial dysfunction, cancer cells face challenges in obtaining nutrients and energy supply. However, monounsaturated fatty acids have been identified as preferred substrates for energy generation and maintenance of cellular structures in cancer cells. These fatty acids can be obtained through de novo fatty acid synthesis. Stearoyl-CoA desaturase 1 (SCD1) is the primary enzyme involved in the biosynthesis of monounsaturated fatty acids from saturated fatty acids and is known to be upregulated in various malignancies, including breast cancer. This study aimed to assess the expression and activity index of SCD1 in well-characterized MCF-7, SK-BR3, and MDA-MB231 breast cancer cell lines, representing luminal A/B, HER2-enriched, and triple-negative subtypes, respectively. Methods The breast cancer cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity for 72 hours. After harvesting the cells, SCD1 expression was analyzed using real-time quantitative PCR, and the activity index was determined by measuring the ratio of monounsaturated fatty acids to saturated fatty acids using capillary gas-liquid chromatography. Results MCF-7 cell line showed a significantly higher SCD1 expression compared to MDA-MB231 (250-fold, p&amp;lt; 0.0001) and SK-BR3 (83-fold, p&amp;lt; 0.0001) cell lines. The activity index of SCD1 was significantly higher in both MCF-7 and MDA-MB231 cell lines with fold changes of 2.6 and 2.5, respectively, compared to SK-BR3 cell line (p=0.004 for both). Conclusion Cell model assays demonstrated that the luminal A/B and triple-negative subtypes showed a higher capacity to convert saturated fatty acids to monounsaturated fatty acids through SCD1 enzyme activity, indicating a different lipid metabolism profile in the HER2-enriched subtype. These new findings reveal differences in SCD1 expression and activity among breast cancer subclasses. They highlight vulnerabilities in metabolism and identify potential therapeutic targets with clinical implications for managing breast cancer. Citation Format: Hamid-Reza Feizi, Amir Mehdizadeh, Zohreh Sanaat, Khadijeh Abbasi, Frank Gieseler, Masoud Darabi. Comparative analysis of stearoyl-CoA desaturase 1 expression and activity index in model cell lines of breast cancer subclasses [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 PO1-13-08.

The potent N-methyl-d-aspartate (NMDA) receptor antagonists 1-3 have been demonstrated to show antiproliferative and cytotoxic effects in MCF-7 and SKBR3 breast cancer cell lines. To improve the knowledge about the role played by the NMDA receptor in the antitumor activity of these compounds, the enantiomers of 1 were prepared and evaluated for their affinity for the phencyclidine (PCP) site of the NMDA receptor and for their cytotoxic effect in MCF-7 and SKBR3 cell lines, both expressing the NMDA receptor. The (S)-1 enantiomer, showing negligible affinity for the PCP site, exhibited antiproliferative activity higher than that of (R)-1, which instead bound the PCP site. The downregulation of NMDA GluN1 expression resulted in a decreased (S)-1-induced cytotoxicity and apoptotic cell death, unequivocally demonstrating the involvement of the NMDA receptor in the antitumor effect of this compound. Due to its interesting biological profile, (S)-1 represents a lead compound to develop novel antitumor agents for breast cancer treatment.

PKCδ and MAPK mediate G 1 arrest induced by PMA in SKBR-3 breast cancer cells

Hesperetin (HSP), a flavonoid, has been validated to modify gene expression and function as an epigenetic agent to stop the development of breast carcinoma cells. HSP was investigated in this research to evaluate the expression of the MLH1 and MSH2 genes in cancerous breast cell lines (SKBR3) and healthy cell lines (MCF-11A) after exposure to different dosages (200, 400, and 600 µM/mL) of HSP. After 48 h of exposure, SKBR3's half-maximal inhibitory concentration was 289.6 µM/mL and MCF-10A's was 855.4 µM/mL. The research found that increasing HSP concentrations were closely correlated with an increase in MLH1 gene levels in the SKBR3 cell line, as shown by median and percentile values. HSP therapy caused the MLH1 gene expression to substantially vary in different groups, and in the SKBR3 cell line, MSH2 gene expressions were elevated in a dose-escalating manner. Moreover, HSP also raised the number of apoptotic cells, with the fraction of apoptotic cells escalating substantially at doses of 400 and 600 µM/mL. The outcomes suggested that HSP has the potential to be utilized as a therapeutic intervention for breast cancer, as it can induce apoptosis and reduce cell viability.

Objectives: HER2 over-expression in breast cancer is associated with a poor prognosis and HER2 is an intense target of immunotherapy with both humoral and T cell -base approaches. HER2 expression is also known to facilitate the escape of tumor cells from immune surveillance by down-regulating MHC class I, resulting in a reduced sensitivity to CTL lysis. Restoring tumor cell MHC Class I expression and reversing their immune evasion are therefore important strategies in the design of immunotherapy against HER2 expressing tumors. In this study we investigated the effects of monoclonal antibody trastuzumab and T help-1 associated cytokines IFN-γ and TNF-A on HLA Class I expression and the susceptibilities to CTL lysis of human HER2 expressing tumor cell lines. Methods: Breast cancer cell lines MCF7, SKBR3, BT474 and ovarian cancer cell line SKOV3 were treated with trastuzumab only, combination of IFN-γ and TNF-A or trastuzumab with IFN-γ and TNF-A. Cell surface expressions of HLA ABC were examined by flow cytometric analysis. The susceptibility of tumor cells to HER2 specific CD8+ T cell recognition and CTL lysis were assessed by measurement of CD8+ T cell IFN-γ secretion and flow cytometric analysis respectively. Results: Th1 cytokines IFN-γ and TNF-A dramatically increased HLA ABC expressions on all tumor lines compared to untreated cells, [ MCF7 (p=0.04); SKBR3 (p=0.02); SCOV3 (p=0.02) and BT474 (p=0.04)], while trastuzumab alone showed little impact on HLA ABC up-regulation. When treated with a combination of trastuzumab, IFN-γ and TNF-A tumor cells displayed synergistically enhanced HLA ABC expression [MCF7 (p=0.02); SKBR3 (p=0.004); SKOV3 (0.006) and BT474 (p=0.03)]. We observed that IFN-γ and TNF-A treatment was able to remarkably increase CD8+ T cell recognition and CTL lysis of low and intermediate HER2 expressing tumors [ MCF7(p=0.03) and SKOV3(0.02)], but not High HER2 tumor[ SKBR3(p=0.07)]. Only a combined treatment of trastuzumab, IFN-γ and TNF-A rendered SKBR3 susceptible to a significant killing by CTL (p=0.02). To investigate the impact of other HER family members EGFR and HER3 on MHC class I expression , HER2 over-expressing breast cancer cell lines BT474 and SKBR3 were pre-treated with EGF or Heregulin, then subjected to IFN-γ and TNF-A treatments. Activation of HER1 and HER3 signaling made HER2 over-expressing cells resistant to the effect of MHC class I up-regulation by IFN-γ and TNF-A. Conclusion: Both humoral and cellular immune HER-2 targeted interventions are required to reverse immune escape mediated by HER2; in addition to targeting HER2, targeting HER1 and HER3 signaling elements should also be included in order to abrogate the cross talk between HER family members and more effectively inhibit the HER2 signaling pathway. Citation Format: Shuwen Xu, Jessica Cintolo, Jashodeep Datta, Cinthia Rosemblit, Erik Berk, Julia Terhune, Elizabeth Fitzpatrick, Brian Czerniecki. Reversal of immune evasion mediated by HER2 requires both humoral and cellular HER-2 targeted immune interventions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2014-4079

Objective: To investigate the effect of long non-coding RNA-AC013472.3 on lipopolysaccharide (LPS)-stimulated secretion of tumor necrosis factor (TNF)-α in NR8383 rat alveolar macrophages. Methods: Silencing and overexpression models of lncRNA-AC013472.3 were established with NR8383 rat alveolar macrophages as the experimental subjects. The silencing models were divided into three groups: random nonsense negative small interfering RNA sequence (si-con) group (si-con group, si-con transfected NR8383 cells), LPS+si-con group (10 μg/L LPS was used to treat si-con transfected NR8383 cells for 24 h), and siRNA group (siRNA transfected NR8383 cells), and LPS+siRNA group (10 μg/L LPS was used to treat siRNA transfected NR8383 cells for 24 h). The overexpression models were divided into the empty plasmid (p-con) group (p-con transfected NR8383 cells), LPS+p-con group (10 μg/L LPS was used to treat p-con transfected NR8383 cells for 24 h), lncRNA overexpression plasmid (plncRNA) group (plncRNA transfected NR8383 cells), and the LPS+plncRNA group (10 μg/L LPS was used to treat plncRNA transfected NR8383 cells for 24 h). The mRNA levels of TNF-α in each group were examined by quantitative real-time PCR (qPCR). The protein levels of tumor necrosis factor receptor-related factor-6 (TRAF-6) and phosphorylated nuclear factor-κB (NF-κB) p65 were examined by Western blot. Results: In the silencing model, the mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+si-con group were significantly higher than those in the si-con group (2.040±0.195 vs 1.048±0.207, 0.473±0.022 vs 0.293±0.076 and 0.469±0.062 vs 0.252±0.038)(all P<0.05). The mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+siRNA group were significantly higher than those in the siRNA group (4.158±0.119 vs 1.028±0.019, 0.700±0.104 vs 0.231±0.023 and 0.771±0.095 vs 0.258±0.050)(all P<0.05). The relative expression levels of all indexes in the LPS+siRNA group were significantly higher than those in the LPS+si-con group (all P<0.05). In the overexpression model, the mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+p-con group were significantly higher than those in the p-con group (1.961±0.169 vs 0.999±0.143, 0.533±0.047 vs 0.247±0.020 and 0.565±0.108 vs 0.276±0.048) (all P<0.05). The mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+plncRNA group were significantly higher than those in the plncRNA group (1.322±0.110 vs 1.043±0.093, 0.347±0.035 vs 0.232±0.023 and 0.405±0.072 vs 0.268±0.031) (all P<0.05). The relative expression of all indexes in the LPS+plncRNA group were significantly lower than that in the LPS+p-con group (all P<0.05). Conclusion: LncRNA-AC013472.3 may inhibit the activation of NF-κB signaling pathway, thereby inhibiting the LPS-stimulated secretion of TNF-α in NR8383 rat alveolar macrophages.

Introduction: Treatment strategies for HER2+ breast cancer (BC) involves first line treatment with the monoclonal antibody trastuzumab, however resistance to treatment can occur. The pan-HER tyrosine kinase inhibitor neratinib is an approved adjuvant treatment for early stage HER2+ BC after one year of treatment with trastuzumab. Recent studies have highlighted the potential benefits of using retinoic acid as an adjuvant treatment for BC. The retinoic acid receptor (RAR) family of nuclear transcription factors consists of RARα, RARβ and RARγ. Interestingly ~30% of HER2+ BC have a co-amplification of the RARA gene, which codes for RARα. We examined the effect of RARα agonists (Fenretinide &amp; AM580) and antagonist (AGN194310) in combination with neratinib in two HER2+ cell lines: SKBR3, has co-amplification of ERBB2 and RARA, and HCC1569 has amplified ERBB2 but are not RARA-amplified. Methods: SKBR3 and HCC1569 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. 10mM stock concentrations of fenretinide (H7779-Sigma), AM580 (A8843-Sigma), AGN194310 (SML2665-Sigma) and neratinib (Puma Biotechnology, Inc) were prepared in DMSO. For proliferation assays, cells were seeded in 96 well plates at a density of 3 X 104 cells per well for 24h. Cells were treated with a drug alone (2X concentration) or combination of drugs (4X concentration) in 100μL of medium. Proliferation was measured using an acid phosphatase-based assay after 5 days as percentage growth versus DMSO control. The half maximal inhibitory concentration (IC50) was calculated for each drug, using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50), using CalcuSyn. Values &amp;lt; 1 represent a synergistic effect, a value of 1 is additive and values &amp;gt; 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research has found that the synthetic retinoic acid fenretinide, a pan-RAR (α, β and γ) activator, was more potent in the HCC1569 cells compared to the SKBR3 cell line with an IC50 of 0.21 ± 0.03μM compared to 4.55 ± 0.87 μM, respectively. However, when combined with neratinib there was a strong antagonistic effect observed in the HCC1569 cell line (CI value: 15.63 ± 9.5). Conversely, fenretinide enhanced the effect of neratinib in the SKBR3 cell line (CI value: 0.83 ± 0.4). The RARα-specific activator AM580 had an IC50 &amp;gt;10μM for both cell lines. When combined with neratinib there was an additive effect observed in the RARα negative HCC1569 cells (CI value: 0.97 ± 0.4), and a synergistic effect observed in the RARα upregulated SKBR3 cell line (CI value: 0.78 ± 0.08). Next we wanted to determine if inhibiting RAR activity would have any effect on the proliferation of these cell lines. The IC50 for the pan-RAR (α, β and γ) antagonist AGN194310 was &amp;gt;10μM for each cell line. However, when combined with neratinib treatment there was a strong synergistic effect observed in the HCC1569 cell line (CI value: 0.52 ± 0.17) and the SKBR3 cell line (CI value: 0.66 ± 0.19). Conclusions: This study suggests a differential effect of RAR agonists when combined with neratinib in RARα amplified versus non-amplified HER2+ BC. Targeting RAR signalling, particularly with a RAR antagonist, in combination with the pan-HER inhibitor warrants further investigation in HER2+ BC. Citation Format: Debbie O'Reilly, Nicola Gaynor, Neil Conlon, Irmina Diala, Lisa D Eli, John Crown, Denis M Collins. Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines [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 P2-13-28.

Objective The purpose of this study was to develop multivalent antibody constructs via grafting anti-HER2 antibodies, including Herceptin and oligoclonal-variable domain of heavy chain antibodies (VHHs), onto liposome membranes to enhance antibody activity and compare their effect on phospholipase C (PLC) signaling pathway with control. Materials and Methods In this experimental study, SKBR3 and BT-474 cell lines as HER2 positive and MCF10A cell line as normal cell were screened with anti-HER2 antibodies, including constructs of multivalent liposomal antibody developed with Herceptin and anti-HER2 oligoclonal-VHHs. To confirm the accuracy of the study, immunofluorescent assay, migration assay and immuno-liposome binding ability to HER2 were evaluated. Finally, the antibodies effect on PLCγ1 protein level was measured by an immunoassay method (ELISA). Results In the present study, by using multivalent form of antibodies, we were able to significantly inhibit the PLCγ1 protein level. Interestingly, the results of migration assay, used for study the motility of different types of cell, shows correspondingly decreased number of immigrated cells in SKBR3 and BT-474 cell lines. Since MCF10A cells show no overexpression of HER2, as expected, the result did not show any change in PLCγ1 level. Moreover, immunofluorescent assay has confirmed high expression of HER2 in SKBR3 and BT-474 cell lines and low HER2 expression on MCF10A cell line. High binding of immuno-liposome to SKBR3 and BT-474 cells and low binding to MCF10A confirmed that in this study anti-HER2 antibodies have conserved binding ability to HER2 even after conjugation with liposome. ConclusionPLCγ1 protein levels did indeed decrease after treatment with immuno-liposome form of compounds in both two tested cell lines, verifying the inhibition ability of them. Moreover, an elevated antibody activity is associated with liposomes conjugation suggesting that immuno-liposome may be a potential target for enhancing the antibody activity.