BT474 Cell Lines Research Articles

Dolutegravir Inhibits Proliferation and Motility of BT-20 Tumor Cells Through Inhibition of Human Endogenous Retrovirus Type K.

Increasing evidence points to the role of endogenous retroviruses (ERVs) in driving cancer cell proliferation. The purpose of this study was to explore the possibility of repurposing antiretroviral agents to inhibit ERVs as a new approach to cancer treatment. We found that an integrase strand-transfer inhibitor, dolutegravir (DTG), effectively inhibited the proliferation of multiple cancer cell lines and its antiproliferative potency was positively correlated with the expression levels of the human endogenous retrovirus type K (HERV-K). DTG inhibited the expression of HERV-K in multiple human cancer cell lines and the mouse mammary tumor virus (MMTV) in the murine 4T1 mammary cancer cell line. We chose the fast-growing BT-20 cell line as a model to study the in vitro antiproliferative mechanisms of DTG. BT-20 cells overexpressing both HERV-K env and pol genes became more resistant to DTG than cells transduced with vector alone. Knockdown of HERV-K also increased DTG resistance of BT-20 cells. The antiproliferative effect of DTG correlated with enhanced expression of E-cadherin and reduction in cell motility and invasiveness. Surprisingly, DTG stimulated expression of the env gene of MMTV in vivo and promoted metastasis of 4T1 tumor cells to the lungs. Taken together, our data support the role of ERVs in tumor development and encourage the further search for antiretroviral agents to treat malignancies in which ERVs are active.

High Expression of p62 and ALDH1A3 Is Associated With Poor Prognosis in Luminal B Breast Cancer.

p62 (also known as sequestosome 1) is involved in cancer progression, and high expression of p62 indicates poor clinical outcome in several cancer types. However, the association between p62 gene expression and cancer stem cells (CSCs) in breast cancer subtypes remains unclear. In the present study, genomic datasets of primary breast cancer (The Cancer Genome Atlas, n=593; and Molecular Taxonomy of Breast Cancer International Consortium, n=2,509) were downloaded. p62 Expression was then examined in normal and breast cancer tissues derived from the same patients. Kaplan-Meier and multivariate Cox regression analyses were employed to evaluate disease-specific survival. Next, the effect on cell viability and in vitro tumor-sphere formation of p62 knockdown using targeted small interfering RNA was assessed by using cells with high activity of aldehyde dehydrogenase 1 (ALDH1high). Patients with normal-like, luminal A or luminal B breast cancer with p62high had poor prognosis. Furthermore, patients with p62high ALDH1A3high luminal B type also exhibited poor prognoses. Knockdown of p62 suppressed viability and tumor-sphere formation by ALDH1high cells of the luminal B-type cell lines BT-474 and MDA-MB-361. These results suggest that p62 is essential for cancerous progression of ALDH1-positive luminal B breast CSCs, and contributes to poor prognosis of luminal B breast cancer. p62 is potentially a prognostic marker and therapeutic target for ALDH1-positive luminal B breast CSCs.

Thymoquinone upregulates IL17RD in controlling the growth and metastasis of triple negative breast cancer cells in vitro

BackgroundTriple negative breast cancer (TNBC) is a molecular subtype of breast cancer, which is a major health burden of females worldwide. Thymoquinone (TQ), a natural compound, has been found to be effective against TNBC cells, and this study identified IL17RD as a novel target of TQ in TNBC cells.MethodsWe have performed chromatin immunoprecipitation Sequence (ChIP-Seq) by MBD1 (methyl-CpG binding domain protein 1) antibody to identify genome-wide methylated sites affected by TQ. ChIP-seq identified 136 genes, including the tumor suppressor IL17RD, as a novel target of TQ, which is epigenetically upregulated by TQ in TNBC cell lines BT-549 and MDA-MB-231. The IL17RD expression and survival outcomes were studied by Kaplan–Meier analysis.ResultsTQ treatment inhibited the growth, migration, and invasion of TNBC cells with or without IL17RD overexpression or knockdown, while the combination of IL17RD overexpression and TQ treatment were the most effective against TNBC cells. Moreover, higher expression of IL17RD is associated with longer survival in TNBC patients, indicating potential therapeutic roles of TQ and IL17RD against TNBC.ConclusionsOur data suggest that IL17RD might be epigenetically upregulated in TNBC cell lines by TQ, and this might be one of the mechanisms by which TQ exerts its anticancer and antimetastatic effects on TNBC cells.

Abstract 189: Evaluating the influence of Lactobacillus rhamnosus-derived extracellular vesicles on calcium deposition in early breast cancer

Abstract Microcalcifications are an important mammographic feature of ductal carcinoma in situ (DCIS) and are detected in 85-90% of abnormal mammograms. Calcium transport and calcium channels regulate microcalcifications and are essential for breast cancer proliferation. The breast duct intraductal lumen, which is the origin of ductal carcinomas, has been shown to be a non-sterile environment. We hypothesize that the intraductal microbiome influences calcium handling and mineral deposition in breast cancer. Bacteria have been known to enhance the formation of mineral deposits, including calcium salts, in various non mammalian microenvironments via secreted factors or extracellular vesicle mediated nucleation. We performed a clinical study on a cohort of 150 women who received a suspicious mammogram and were subjected to a follow up biopsy, and we identified differences in calcium handling microbiota between patients who harbored a malignant disease with respect to benign controls. To mechanistically study the role of microbiota in breast cancer calcium handling, we developed a 3D spheroid model of ductal carcinoma in situ using the BT-474 cell line. When exposed to high concentrations of calcium in the culture medium (from 1.75 mM to 2.05 mM), spheroids developed calcium mineral deposits in a calcium dose dependent manner; deposits were identified in internal regions of the spheroid, which correspond to hypoxic areas of DCIS in vivo. Extracellular calcium deposition correlated with cell survival in face of a toxic calcium level. Spheroids were cultured under a continuous flow of Lactobacillus rhamnosus LMS2-1-derived EVs over the course of two weeks. EVs were isolated by sequential ultracentrifugation; the 100K fraction was retained and characterized in terms of size, concentration, and protein content. EV treatment induced increased calcium export and mineral deposition in a dose dependent manner. Our findings support the hypothesis that L. rhamnosus-derived extracellular vesicles influence calcium regulation and mineral deposition in the hypoxic breast cancer milieu. More studies will be undertaken in the future to unveil molecular mechanisms that underlie this interaction, both at the cancer cell and the microbiota level. Citation Format: Ngoc Bao Vuong, Melany Alomia, Intisar Alruwaili, Fahad Alsaab, James L. Erickson, Weidong Zhou, Alessandra Luchini, Monique Van Hoek, Virginia Espina, Richard A. Hoefer, Lance Liotta. Evaluating the influence of Lactobacillus rhamnosus-derived extracellular vesicles on calcium deposition in early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 189.

Abstract 5653: Increased expression of brain-specific serine/threonine-protein kinase BRSK2 contributes to breast cancer survival by enhancing autophagy under nutrient stress

Abstract Aberrant expression of certain cancer-promoting genes is the associative factor of metastasis and cancer-related death. Autophagy is a highly conserved catabolic process that can be regulated by starvation or stress. Among several cellular stresses, hypoxia and nutrient stress can strongly activate autophagy-related genes. Autophagy plays dual roles in cancer; it can be pro-death or pro-survival. The ability of the cancer cells to survive long-term under stress is still a vast question to cancer researchers. The nutrient status of is sensed by LKB1 downstream kinases AMP-activated protein kinase (AMPK). Recent studies revealed that BRSK2 is one of the AMPK related kinases regulated by LKB1 is the activator of WIPI protein function as building blocks for the formation of the autophagosome. Brain-specific kinases 1 and 2 are highly expressed in the mammalian forebrain regulating neuronal polarity and have lower expression in the testis and pancreas. We identified BRSK2 as one of the top genes highly expressed in 10 primary breast cancer tissue with a record of future bone metastasis compared to 10 primary breast tumors with no metastasis record through RNA seq analysis. Our extended analysis using TCGA and METABRIC cohort data unfolded that BRSK2 is highly elevated in breast cancer tumors compared to control breast tissue (p< 0.001), mainly in stage IV patients compared to other stages (p= 0.01). Furthermore, we observed that BRSK2 is differentially expressed in various breast cancer cell lines, in contrast, no expression in nonmalignant breast epithelial cells (MCF10A). We selected MDA-231 and BT474 cell lines having relatively lower BRSK2 expression, and expressed BRSK2 ectopically to understand details about BRSK2-mediated autophagy under nutrient stress. Interestingly, we observed that BRSK2 augmented nutrient starvation mediated autophagic flux in Triple-negative and ER/PR-positive breast cancer cell lines, along with elevated AKT, p-NF-kB, and STAT3 signaling pathways for survival under nutrient stress conditions. We further observed that several inflammatory cytokines CXCL1, CXCL6, TNF-α, and IL1-β, were significantly upregulated with BRSK2 overexpressed cells compared with the control cells. Knockdown of BRSK2 reduces autophagy and enhances apoptosis in breast cancer cells. Altogether, our data revealed that BRSK2 play a role in increasing autophagy to support cell survival under nutrient stress condition through AKT signaling pathways. Blocking of BRSK2 with inhibitor has reduced autophagy and increased apoptosis and cell death. Thus, BRSK2 can be a potential molecule as a therapeutic target for breast cancer metastasis. Citation Format: Nitai Hait, APARNA MAITI, Rongrong Wu, Kazuaki Takabe, Masanori Oshi. Increased expression of brain-specific serine/threonine-protein kinase BRSK2 contributes to breast cancer survival by enhancing autophagy under nutrient stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5653.

Abstract 5827: Increased miR150 expression in tumors enhances lymphocytes infiltration causing immunosuppression through inflammatory chemokines

Abstract Dysregulated miRNAs play key roles in multiple biological processes and cancer. miR-150-5p, a cancer-related miRNA, has been reported to be aberrantly expressed in various cancers. We found that triple-negative and HER2+ breast cancer patients have elevated expression of miR150 in the TCGA and METABRIC patient cohort. Both cohorts showed significantly increased expression of miR150 in grade IV patients. Further studies explained that miR150 is strongly correlated with CD8+ T cells and B cells in accord with increased expression of immune-checkpoint markers PD-1, PDL-1, and CTLA4. We, therefore, hypothesized that higher expression of miR150 in tumors attracted more immune cells in the tumor microenvironment. We use two triple negative breast cancer cell lines MDA-231 and BT-549 cells and overexpressed miR150 followed by co-culture them with Jurkart and B cells. Our data revealed that both jurkart and B cells had increased migration towards cancer cells overexpressed miR150 mimic compared to miR150 mimic negative control. Interestingly, overexpressed miR150 mimic in cancer cells inhibited migration in both triple-negative breast cancer cells. Overexpression of miR150 mimic in cancer cells did not alter cell proliferation. To understand further the role of miR150 in breast cancer, we overexpressed miR150 mimic and its corresponding negative control in MDA-231 and did RNA seq analysis. RNA seq data explained that approximately 4927 genes are upregulated with miR150 mimic overexpression. Interestingly several chemokines and mediators of metastasis (CXCL2, CXCL1 CCL20, IGBP3, and ANGPTL4) are upregulated among the top 50 upregulated genes. In addition, gene enrichment analysis explained that TNF-α signaling pathways, Hypoxia, epithelial-mesenchymal pathways, E2F signaling, and Inflammatory pathways are a few upregulated pathways with miR150 mimic overexpression compared to the negative control. We also found miR150 high tumor enriched correlated with interferon-alpha and gamma signaling, inflammatory response pathways similar with cohort analysis data. In conclusion, miR150 in cancer plays a key role in interferon and inflammatory signaling pathways attracting more immune cells and suppressing the tumor microenvironment. Citation Format: Aparna Maiti, Masanori Oshi, Rongrong Wu, Nitai Hait, Kazuaki Takabe. Increased miR150 expression in tumors enhances lymphocytes infiltration causing immunosuppression through inflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5827.

Abstract 5290: BSI-001, a novel anti-HER2 antibody exhibiting potent synergistic efficacy with trastuzumab

Abstract Background: Trastuzumab and pertuzumab are anti-HER2 antibodies that bind to two different epitopes of HER2. Although pertuzumab itself did not exhibit substantial clinical benefit as monotherapy, it was approved to be used in combination with trastuzumab because of its ability to enhance the clinical efficacy of trastuzumab. Currently, the combination of trastuzumab and pertuzumab is being used as first-line treatment for HER2-positive metastatic breast cancer. However, while the combination of trastuzumab and pertuzumab has been effective, there is still opportunity for greater effectiveness by identifying anti-HER2 mAbs that will be better synergistic partners of trastuzumab, allowing for better efficacy and/or broader indications. Methods: A SynAb࣪ Platform was used to identify BSI-001, an anti-HER2 mAb, through trastuzumab-based synergistic functional screening. A competitive ELISA was used to perform epitope binning of the anti-HER2 antibodies. BSI-001 was further characterized in combination with trastuzumab for its bioactivity in vitro, including inhibition of cell proliferation and antigen-mediated internalization of HER2 positive cell lines. The synergistic effect of BSI-001 with trastuzumab was also evaluated in trastuzumab-resistant cell lines. Multiple animal models were then employed to investigate the synergistic antitumor activity of the combination in vivo. Results: BSI-001 is the best functional partner of trastuzumab identified through the SynAb࣪ platform. BSI-001 binds to a unique epitope, distinct from the binding epitopes of trastuzumab and pertuzumab. It exhibits superior bioactivity and efficacy in vitro and in vivo when combined with trastuzumab. The combination of BSI-001 and trastuzumab inhibits HER2-positive cancer cell growth more potently than that of trastuzumab and pertuzumab. The combination of BSI-001 with trastuzumab also shows inhibition of cell growth of the trastuzumab-resistant cell line BT-474 Clone 5, whereas the combination of pertuzumab and trastuzumab did not. In addition, BSI-001 is able to synergize with trastuzumab to promote antigen-antibody internalization. In animal models, BSI-001 demonstrates superior synergistic antitumor activity when combined with trastuzumab as compared to pertuzumab. Summary: The combination of BSI-001 and trastuzumab showed significantly better in vitro and in vivo activity than the combination of trastuzumab and pertuzumab. In addition, because of the activity of the BSI-001/trastuzumab combination in a trastuzumab-resistant cancer cell line, there is the potential for this combination to be used to treat patients with trastuzumab-resistant cancer or patients who experienced relapse following trastuzumab and pertuzumab treatment. Citation Format: Shukai Xia, Zeyu Peng, Xiaoyu Ding, Wanjian Gu, Yujie Zhong, Jinyu Liu, Hongyan Li, Xiaoyao Hao, Xiaodong F. Liu, Mark Z. Ma, Lan Luo, Chunni Zhang, Mingjiu Chen. BSI-001, a novel anti-HER2 antibody exhibiting potent synergistic efficacy with trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5290.

Abstract 3127: Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod

Abstract Background: Taxanes are extensively used in the chemotherapy of breast cancer in the neoadjuvant, adjuvant and metastatic settings. Paclitaxel is known to have both immunostimulatory and immunosuppressive effects, but its impact on the chemokine system within the tumor microenvironment (TME) remains unclear. In the breast TME treated with neoadjuvant taxane chemotherapy, high levels of the cytotoxic T cell (CTL) attractants CXCL9, CXCL10 and their common receptor CXCR3 predict higher probability of pathological complete response (pCR) and improved long term outcomes. We previously developed a chemokine modulatory regimen (CKM) combining interferon-α with TLR3 agonist rintatolimod to selectively increase CTL-attracting chemokines in the TME but reduce the production of chemokines attracting myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the TME. Here we evaluated paclitaxel’s impact on chemokine production in the human breast TME and CKM’s ability to eliminate undesirable aspects of taxane chemotherapy. Methods: Fresh breast cancer tissues obtained during routine surgeries, breast cancer cell lines BT-549 and MDA-MD-231 and peripheral blood monocyte-derived macrophages were analyzed immediately or cultured ex vivo for 24 hours in the absence or presence of paclitaxel and/or CKM components. The expression of chemokine genes and secretion of chemokines by freshly harvested and ex vivo-treated tumor explants, cancer cell lines and macrophages were analyzed by quantitative PCR (Taqman) and ELISA. Paired student t-test was used for statistical analyses. Results: Breast cancer explants spontaneously expressed high levels of MDSC/Treg-attractants CXCL12 and CCL22, but only marginal levels of CTL and natural killer (NK) cell attractants CXCL9, CXCL10, CXCL11 and CCL5. Unexpectedly, paclitaxel treatment resulted in further elevation of granulocyte/MDSC-attractant CXCL8 and CCL22, which was reversed by the combination of paclitaxel with CKM. At the same time, while paclitaxel alone did not induce any of the CTL attractants tested, its combination with the CKM was highly effective in inducing CXCL9, CXCL10, CXCL11 and CCL5. Conclusions: Our results identify an undesirable aspect of paclitaxel’s impact on breast cancer. The ability of CKM to enhance the expression of CTL/NK cell attractants and suppress the production of Treg/MDSC attractants produced by paclitaxel provides a strong rationale for combined use of CKM in taxane-based chemo-immunotherapy of breast cancer and potentially other diseases. Citation Format: Shipra Gandhi, Melissa Grimm, Ronald Slomba, Kazuaki Takabe, Pawel Kalinski. Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3127.

Cordycepin Inhibits Triple-Negative Breast Cancer Cell Migration and Invasion by Regulating EMT-TFs SLUG, TWIST1, SNAIL1, and ZEB1.

Cancer metastasis is the most important cause of cancer-related death, and epithelial-to-mesenchymal transition (EMT) plays crucial roles in cancer metastasis. Cordycepin (CD) is highly enriched in the medicinally used Cordyceps mushroom. In this study, we conducted the antimetastatic activities of CD, specifically focusing on its regulatory effects on EMT-inducing transcription factors (EMT-TFs) in triple-negative breast cancer (TNBC). Our study showed CD to inhibit the growth, migration, and invasion of BT549 and 4T1 cancer cell lines, by employing cell viability assay and real-time cell analyses. The protein levels of N-Cadherin and E-Cadherin, as well as their transcription factors TWIST1, SLUG, SNAIL1, and ZEB1 in BT549 and 4T1 cells, were estimated by Western blot assays. Results from dual-luciferase reporter assays demonstrated that CD is capable of inactivating the EMT signaling pathway by inhibiting TWIST1 and SLUG expression. Furthermore, in vivo studies with mice carrying cancer cell-derived allograft tumors showed the inhibitory effect of CD on cancer cell growth and metastasis. Furthermore, the additive/synergistic anti-metastasis effect of CD and thymoquinone (TQ), another natural product with promising anticancer roles, was demonstrated by combinational treatment. The results from this research indicate that CD would be a promising therapeutic molecule against TNBC by targeting EMT-TFs, possibly in SLUG, TWIST1, SNAIL1, and ZEB1.

Antagonistic Pharmacological Interaction between Sirtuin Inhibitor Cambinol and Paclitaxel in Triple-Negative Breast Cancer Cell Lines: An Isobolographic Analysis.

Breast cancer (BC) is a heterogeneous disease with different intrinsic subtypes. The most aggressive subtype of BC–triple-negative breast cancer (TNBC) is characterized by high heterogeneity and metastasis rate, poor prognosis and lack of therapeutic targets due to the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Targeted therapies have been approved for many other cancers and even other subtypes of BC, but treatment options for TNBC are still mainly limited to chemotherapy. Therefore, new, more effective treatment regimens are needed. Combined chemotherapy with two or more active agents is considered a promising anti-neoplasm tool in order to achieve better therapeutic response and reduce therapy-related adverse effects. The study demonstrated an antagonistic effect commonly used in TNBC therapy cytostatic drug-paclitaxel (PAX) and sirtuin inhibitor: cambinol (CAM) in BT-549, MDA-MB-468 and HCC1937 TNBC cell lines. The type of pharmacological interaction was determined by a precise and rigorous pharmacodynamic method-isobolographic analysis. The cytotoxic and anti-proliferative effects of CAM used alone or combined with PAX were determined utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2′-deoxyuridine (BrdU) assays, respectively. Induction of apoptosis in TNBC cell lines after PAX and CAM treatment applied individually or in combination was determined by flow cytometry (FACS) as a number of cells with active caspase-3. It has been observed that both agents used separately inhibit cell proliferation and induce apoptosis; however, applying them in combination ameliorated antiproliferative and pro-apoptotic effects in all analyzed TNBC cell lines. Our results demonstrate that CAM and PAX used in combination act antagonistically, limiting anti-cancer efficacy and showing the importance of preclinical testing.

Targeting MDK Abrogates IFN-γ-Elicited Metastasis inCancers of Various Origins.

IFN-γ is a pleiotropic cytokine with immunomodulatory and tumoricidal functions. It has been used as an anti-tumor agent in adjuvant therapies for various cancers. Paradoxically, recent advances have also demonstrated pro-tumorigenic effects of IFN-γ, especially in promoting cancer metastasis, with the mechanism remains unclear. This will undoubtedly hinder the application of IFN-γ in cancer treatment. Here, we verified that IFN-γ treatment led to activation of the epithelial-to-mesenchymal transition (EMT) programme and metastasis in cell lines of various cancers, including the kidney cancer cell line Caki-1, the lung cancer cell line A549, the cervical carcinoma cell line CaSki, the breast cancer cell line BT549 and the colon cancer cell line HCT116. We further disclosed that midkine (MDK), an emerging oncoprotein and EMT inducer, is a common responsive target of IFN-γ in these cell lines. Mechanistically, IFN-γ upregulated MDK via STAT1, a principle downstream effector in the IFN-γ signalling. MDK is elevated in the majority of cancer types in the TCGA database, and its overexpression drove EMT activation and cancer metastasis in all examined cell lines. Targeting MDK using a specific MDK inhibitor (iMDK) broadly reversed IFN-γ-activated EMT, and subsequently abrogated IFN-γ-triggered metastasis. Collectively, our data uncover a MDK-dependent EMT inducing mechanism underlying IFN-γ-driven metastasis across cancers which could be attenuated by pharmacological inhibition of MDK. Based on these findings, we propose that MDK may be used as a potential therapeutic target to eliminate IFN-γ-elicited pro-metastatic adverse effect, and that combined MDK utilization may expand the application of IFN-γ in cancer and improve the clinical benefits from IFN-γ-based therapies.

AZD4547 and calcitriol synergistically inhibited BT-474 cell proliferation while modified stemness and tumorsphere formation

Fibroblast growth factor receptor (FGFR) overamplification/activation in cancer leads to increased cell proliferation. AZD4547, a FGFR selective inhibitor, hinders breast cancer cells growth. Although luminal B breast tumors may respond to chemotherapy and endocrine therapy, this subtype is associated with poor prognosis, inadequate response and/or acquired drug resistance. Calcitriol, the vitamin D most active metabolite, exerts anti-neoplastic effects and enhances chemotherapeutic drugs activity. In this study, we sought to decrease the concentration of AZD4547 needed to inhibit the luminal-B breast cancer cell line BT-474 proliferation by its combination with calcitriol. Anti-proliferative inhibitory concentrations, combination index and dose-reduction index were analyzed from Sulforhodamine B assays. Western blot and qPCR were used to study FGFR molecular targets. The compound’s ability to inhibit BT-474 cells tumorigenic capacity was assessed by tumorspheres formation. Results: BT-474 cells were dose-dependently growth-inhibited by calcitriol and AZD4547 (IC50 = 2.9 nM and 3.08 μM, respectively). Calcitriol at 1 nM synergistically improved AZD4547 antiproliferative effects, allowing a 2-fold AZD4547 dose-reduction. Mechanistically, AZD4547 downregulated p-FGFR1, p-Akt and tumorsphere formation. Calcitriol also decreased tumorspheres, while induced cell differentiation. Both compounds inhibited MYC and CCND1 expression, as well as ALDH, a stemness marker that positively correlated with FGFR1 and negatively with VDR expression in breast cancer transcriptomic data. In conclusion, the drugs impaired self-aggregation capacity, reduced stemness features, induced cell-differentiation and when combined, synergistically inhibited cell proliferation. Overall, our results suggest that calcitriol, at low pharmacological doses, may be a suitable candidate to synergize AZD4547 effects in luminal B breast tumors, allowing to reduce dose and adverse effects.

Transcriptome profiling and proteomic validation reveals targets of the androgen receptor signaling in the BT-474 breast cancer cell line

BackgroundAccumulating evidence suggests that the androgen receptor (AR) and its endogenous ligands influence disease progression in breast cancer (BCa). However, AR-mediated changes in BCa differ among the various BCa subtypes according to their hormone receptor profile [i.e., presence/absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2, (HER2)]. Thus, we explored the androgen-regulated transcriptomic changes in the ER+PR+HER2+ BCa cell line, BT-474, and compared them with PR-mediated changes.MethodsWe performed RNA sequencing analysis in treated BT-474 cells with dihydrotestosterone (DHT) and progesterone. Validation of the top ten differentially androgen-regulated genes and a number of other genes found in enriched signaling pathways was performed by qRT-PCR in BT-474 and other BCa cell lines. In addition, a parallel reaction monitoring targeted proteomic approach was developed to verify selected transcripts at the protein level.ResultsIn total 19,450 transcripts were detected, of which 224 were differentially regulated after DHT treatment. The increased expression of two well-known androgen-regulated genes, KLK2 (p < 0.05) and KLK3 (p < 0.001), confirmed the successful androgen stimulation in BT-474 cells. The transcription factor, ZBTB16, was the most highly upregulated gene, with ~ 1000-fold change (p < 0.001). Pathway enrichment analysis revealed downregulation of the DNA replication processes (p < 0.05) and upregulation of the androgen signaling and fatty acid metabolism pathways (p < 0.05). Changes related to progesterone treatment showed opposite effects in gene expression than DHT treatment. Similar expression profiles were observed among other BCa cell lines expressing high levels of AR (ZR75.1 and MBA-MB-453). The parallel reaction monitoring targeted proteomic analysis further confirmed that altered protein expression (KLK3, ALOX15B) in the supernatant and cell lysate of DHT-treated BT-474 cells, compared to control cells.DiscussionOur findings suggest that AR modulates the metabolism of BT-474 cells by affecting the expression of a large number of genes and proteins. Based on further pathway analysis, we suggest that androgen receptor acts as a tumor suppressor in the BT-474 cells.

AXL/PLCγ1/PKCα as a Central Axis for PD‐L1 Regulation in Triple‐Negative Breast Cancer

Breast cancer is the most prevalent malignancy in women globally, accounting for 12% of all new annual cancer cases worldwide. Triple-negative breast cancer (TNBC), which is ER, PR and HER2 negative, represents 10-15% of breast cancers and displays an aggressive metastatic phenotype, with limited treatment options and poor patient prognosis. TNBC is associated with high expression levels of immune checkpoint programmed-death ligand 1 (PD-L1) compared with other breast cancer subtypes, and it is therefore amenable to immune checkpoint therapy. In this study, we analyzed the role of diacylglycerol (DAG)-regulated protein kinase C alpha (PKCα) in the control of PD-L1 expression in TNBC. Dataset analysis of 50 breast cancer cell lines revealed a prominent up-regulation of PKCα in TNBC, particularly in TNBC-B subtype, with a concomitant down-regulation of protein kinase C delta (PKCδ). This finding was authenticated in the TCGA-BRCA database. A significant correlation was found between PKCα expression and EMT markers, i.e., up-regulation of vimentin, Zeb1, TWIST1, SNAI2 and AXL, and E-cadherin down-regulation. High PKCα expression was also observed in TNBC patient-derived xenografts (PDXs). Immunofluorescence analysis revealed significant PKCα endogenous levels in the TNBC B cell line BT549, with strong "peripheral staining", a hallmark of its activated state. Conversely, a tenuous PKCα staining was detected in luminal breast cancer cells. Notably, TNBC-B cell lines express high levels of phosphorylated (active)-phospholipase C gamma 1 (PLCγ1), an enzyme responsible for DAG generation and PKCα activation. We also found a significant positive correlation between PKCα and PD-L1 expression in breast cancer cell lines. Notably, both RNAi silencing and pharmacological inhibition of PKCα or PLCγ1 reduced PD-L1 mRNA and protein levels in TNBC cells. We also show that AXL and PKCα mutually and positively control their expression in addition to up-regulating PD-L1, and they share a common gene expression signature that is highly enriched in cytokine, extracellular matrix, and motility pathways. Mechanistic analysis showed that inhibition of AXL, PLCγ1 or PKCα significantly reduced PD-L1 promoter activity in TNBC cells, suggesting a transcriptional control of the PDL1 (CD274) gene by this pathway. In summary, our results indicate that aberrant overactivation of AXL/PLCγ1/PKCα represents a cancer cell intrinsic mechanism for PD-L1 up-regulation in TNBC, suggesting a potential role for this axis in the control of tumor immunosurveillance. PKCα and AXL may be promising candidates for integrated targeted therapy in TNBC treatment.

Fully Natural Lecithin Encapsulated Nano-Resveratrol for Anti-Cancer Therapy.

IntroductionChemotherapeutics are known to have undesirable side effects (i.e. nausea, weight loss, hair loss, weakened immune system, etc.) due to the non-specificity of the drugs. Encapsulation of these chemotherapeutics inside nanoparticles significantly improves the bioavailability and half-life of drugs, while increasing their tumor penetration and localization. However, most, if not all, nanoparticles in clinics or research are synthetic, with no long-term studies on the effect of these nanoparticles in vivo. Herein, we developed a synergistic resveratrol nanoparticle system by using lecithin encapsulation. Lecithin, being a fully natural phospholipid derived from soybean, possesses inherent anti-tumor activity.MethodsLec(RSV) was successfully prepared using the nanoprecipitation method, and characterized by particle size and zeta potential analysis, and transmission electron microscopy (TEM). The in vitro cellular uptake and cytotoxic effects of Lec(RSV) were investigated in human breast cancer cell line BT474. Finally, the in vivo tumoral uptake of Lec(RSV) was carried out in the BT474 orthotopic model.ResultsLec(RSV) showed a uniform distribution of ~120 nm, with prolonged stability. Lec(RSV) showed high cellular uptake and anti-cancer properties in vitro. Time-dependent uptake in the BT474 xenograft model indicated an increased tumoral uptake and apoptosis rate at 4 hours after tail vein injection of Lec(RSV).ConclusionTaken together, we successfully developed a fully natural Lec(RSV) that possesses potent anti-cancer activity in vitro, with good tumoral uptake in vivo. We hypothesize that Lec(RSV) could be a safe anti-cancer therapeutic that could be easily translated into clinical application.

Palladium-Mediated Incorporation of Carboranes into Small Molecules, Peptides, and Proteins.

Carboranes represent a class of compounds with increasing therapeutic potential. However, few general approaches to readily embed carboranes into small molecules, peptides, and proteins are available. We report a strategy based on palladium-mediated C-X (X = C, S, and N) bond formation for the installation of carborane-containing moieties onto small molecules and peptides. We demonstrate the ability of Pd-based reagents with appropriate ligands to overcome the high hydrophobicity of the carborane group and enable chemoselective conjugation of cysteine residues at room temperature in aqueous buffer. Accordingly, carboranes can be efficiently installed on proteins by employing a combination of a bis-sulfonated biarylphosphine-ligated Pd reagent in an aqueous histidine buffer. This method is successfully employed on nanobodies, a fully synthetic affibody, and the antibody therapeutics trastuzumab and cetuximab. The conjugates of the affibody ZHER2 and the trastuzumab antibody retained binding to their target antigens. Conjugated proteins maintain their activity in cell-based functional assays in HER2-positive BT-474 cell lines. This approach enables the rapid incorporation of carborane moieties into small molecules, peptides, and proteins for further exploration in boron neutron capture therapy, which requires the targeted delivery of boron-dense groups.

Molecular Networking-Based Screening Led to the Discovery of a Cyclic Heptadepsipeptide from an Endolichenic Xylaria sp.

MS/MS-based molecular networking strain prioritization led to the discovery of a group of cyclic depsipeptides from an endolichenic Xylaria sp. The main component, xylaroamide A (1), was obtained by LC-MS-guided isolation. The planar structure of compound 1 was elucidated via 1D and 2D NMR, as well as MS/MS data. The configurations were fully determined by the combination of advanced Marfey's analysis, partial hydrolysis, Mosher's reaction, and GIAO NMR calculation based on a restricted conformational search. A plausible biosynthetic pathway for xylaroamide A (1) involving a rare trans-acting N-methyltransferase is proposed based on bioinformatics analysis. Xylaroamide A (1) exhibited inhibitory activity against cancer cell lines BT-549 and RKO with IC50 values of 2.5 and 9.5 μM, respectively.

GDC-0941 activates integrin linked kinase (ILK) expression to cause resistance to GDC-0941 in breast cancer by the tumor necrosis factor (TNF)-α signaling pathway

ABSTRACT Breast cancer is characterized by high morbidity and mortality. GDC-0941 is a PI3K inhibitor with oncogenic effects in breast cancer. However, certain breast cancer cells are insensitive to GDC-0941. Hence, the mechanism of GDC-0941 in breast cancer resistance was investigated in this study. Breast cancer cell lines BT-474, MCF7, Hs-578-T, MDA-MB-231, MDA-MB-453, and MDA-MB-468 were cultured in different medium and then treated with 100 or 500 nM GDC-0941, 100 nM OSU-T315, or TNF-α antibody. Moreover, ILK and shILK were transfected into cells. The half maximal inhibitory concentrations (IC50) for GDC-0941 were detected using CCK-8 assay. The levels of ILK, AKT, PDK1, S6, and p70S6K expression were detected using western blotting and qPCR. In addition, the mouse model of breast cancer was constructed to measure the tumor size, volume, and weight. The results showed that GDC-0941 decreased cell survival rate, downregulated the phosphorylation of AKT, S6, and p70S6K, and promoted the expression of ILK, while it had little effect on PDK1 expression. GDC-0941 inhibited the increases in p-AKT, p-S6, and p-p70S6K caused by ILK overexpression and promoted ILK knockdown-induced reduction of p-AKT, p-S6, and p-p70S6K. In addition, the combination of OSU-T315 and GDC-0941 decreased p-AKT, p-S6, and p-p70S6K level, tumor volume, and tumor weight. GDC-0941 promoted ILK expression by upregulating TNF-α level. Taken together, GDC-0941 increased ILK level by upregulating TNF-α, thus affecting AKT expression and the sensitivity of breast cancer cells to GDC-0941.

Synergistic Role of Thymoquinone on Anticancer Activity of 5-Fluorouracil in Triple Negative Breast Cancer Cells.

Triple Negative Breast Cancer (TNBC) is considered as the most deadly subtype of breast cancer, because of heterogeneity, less treatment options and resistance to chemotherapy. To find out an efficient chemotherapeutic options, in this study we have investigated the combined therapy of 5-Fluorouracil (5-FU) and thymoquinone (TQ) against TNBC cell lines BT-549 and MDA-MB-231. We have tested 5-FU and TQ alone and in combination (5-FU + TQ) to observe the cellular growth, cell cycle and apoptosis status of BT-549 and MDA-MB-231 cells. Also we have measured the mRNA level expression of genes related to cell cycle and apoptosis. Experimental results suggest that both of 5-FU and TQ are effective in controlling cell growth, cell cycle and inducing apoptosis, but their combination is much more effective. 5-FU was found to be more effective in controlling cell growth, while TQ was found to be more effective in inducing apoptosis, but in both cases, their combination was most effective. TQ was found more effective in increasing and BAX/BCL-2 ratio, while 5-FU was more effective in inhibiting thymidylate synthase. They showed significant increasing effects on caspases and P53 and decreasing effect on CDK-2, where their combination was found most effective. Thus, TQ and 5-FU probably showed synergistic effect on both of cell cycle and apoptosis of tested TNBC cell lines. Our study reveals that TQ can synergise 5-FU action, and increase its anticancer efficiency against TNBC cells, which might be good choice in drug development for TNBC treatment.

Correlation between CA12 and TFF3 and their prediction value of neoadjuvant chemotherapy response in breast cancer.

Compared with other molecular subtypes, hormone receptor-positive breast cancer often shows worse neoadjuvant chemotherapy efficacy. This study aims to explore the relationship between the oestrogen receptor (ER)-related genes carbonic anhydrase 12 (CA12) and trefoil factor 3 (TFF3) and their predictive value of neoadjuvant chemotherapy for breast cancer. We investigated the relationships between CA12, TFF3 and ER status and their predictive value of anthracycline-taxane neoadjuvant chemotherapy in 115 female breast cancer patients via real-time polymerase chain reaction (RT-PCR) and 4 GEO datasets: GSE41998, GSE25065, GSE20194 and GSE20271. Then, the effects of CA12 and TFF3 on the chemotherapy drugs doxorubicin and docetaxel were verified in vitro in the breast cancer cell lines MCF-7 and BT474. The GEO datasets and RT-PCR results showed that the relative expression of both CA12 and TFF3 was higher in oestrogen receptor-positive samples compared with the other samples (p<0.05). CA12 was significantly correlated with TFF3 (p<0.05). In MCF-7 cells, inhibition of TFF3 induced downregulation of CA12 and ESR1 (p<0.05) at both the mRNA and the protein levels, while inhibition of CA12 also downregulated TFF3 and ESR1 (p<0.05). In BT474 cells, inhibition of TFF3 downregulated CA12 and ESR1 (p<0.05) at both the mRNA and the protein levels, while inhibition of CA12led to slight upregulation of TFF3 and ESR1 (p>0.05). Moreover, GEO datasets and RT-PCR results showed that CA12 and TFF3 were more highly expressed in nonpathological complete response (non-pCR) samples than in pCR samples (p<0.05). Cell viability assays of MCF-7 and BT474 cells showed that inhibiting CA12 and TFF3 could enhance sensitivity to doxorubicin and docetaxel (p<0.05). CA12 and TFF3 were correlated with each other, and their high expression might explain the worse efficacy of neoadjuvant chemotherapy in oestrogen receptor-positive breast cancer patients.