Pyrazolidine-3,5-dione Derivatives: Synthesis, Computational, and In Vitro Screening Targeting Breast Cancer

Purpose: The ectopic expression of N6-methyladenosine (m6A) associated genes is a common feature of multiple tumors. However, little is known about the expression status and the prognostic value of these genes in human breast cancer (BRC). Herein, we conducted a comprehensive analysis to identify the expression profiling and clinical significance of m6A-related genomic targets in BRC.Materials and Methods: The expression data including 1109 BRC tissues and 113 normal breast tissues were obtained from The Cancer Genome Atlas (TCGA) database to evaluate the mRNA expression levels of m6A-related genomic targets. In addition, 6 independent BRCA cohorts retrieved from the Gene Expression Omnibus (GEO) database were enrolled to further ascertain the expression profiling of m6A-related genomic targets. Meanwhile, the immunohistochemical (IHC) staining data from BRC tissue microarray (TMA) cohort and the Human Protein Atlas (HPA) database were used to evaluate the proteomic expression of m6A-related genomic targets. Immunofluorescence (IF) analysis was performed to validate the subcellular location of m6A-related genomic targets. Moreover, the prognostic value of m6A-related genomic targets in BRC was analyzed by Kaplan-Meier analysis and Cox regression models.Results: m6A-related genomic targets were differentially expressed in BRC tissues. TMA IHC staining showed that most of the m6A-related genomic targets were significantly altered at the protein level (either upregulated or downregulated), consistent with their changes in the genomic profile. IF analysis showed the subcellular location of m6A-related genomic targets in BRC cell lines. Furthermore, we demonstrated that overexpression of YTHDF1 (P=0.049), YTHDF3 (P<0.001) and KIAA1429 (P=0.032) predicted poor prognosis in terms of overall survival (OS). Upregulation of YTHDF3 was an independent prognostic factor for OS in patients with BRC (P=0.036).Conclusion: m6A-related genomic targets are significantly altered in BRC and predict poor prognosis. These m6A-related genomic targets could serve as novel prognostic biomarkers for BRC.

Introduction - Targeting breast cancer (BC) via its microenvironment is a way to avoid tumor plasticity and development of resistance towards therapy. Bisphosphonates (BPs) are used to treat BC bone metastases. Prior studies suggest that BPs also exert an anti tumor effect by interacting with the microenvironment. However, the precise nature of this interaction is unclear. The chorioallantoic membrane (CAM) assay allows assessment of both human BC and microenvironment cells, circumventing issues of species specificity. Aim - 1) evaluate whether an indirect anti-BC effect by BPs, can be determined using the CAM assay 2) Assess potential biomarkers for this intervention. Methods - MTT survival assays were performed to assess a differential effect of BP zoledronic acid (ZA) on survival of human BC cell lines (MCF-7, SUM149 and MDA-MB-231 progeny cell line SCP2) and human stromal cell line Hs27a. For biomarker analysis, excretion of vascular endothelial growth factor (VEGF)-A and Transforming Growth Factor (TGF) β was measured in the supernatant of Hs27a and MCF-7 cultures and co-cultures treated with ZA by Enzyme-Linked Immuno Sorbent Assays (ELISA). The CAM assay was used to study the effect of ZA on a co-culture system of BC- and stromal cells. SCP2 cells were inoculated on the CAM with and without Hs27a cells on day 6 of embryonic development. ZA (200 μM) or PBS was added on the CAM on day 10 and tumors were harvested at day 14. Immunohistochemistry (IHC) for pan-cytokeratin was performed to identify BC cells in the tumors. Results - ZA (4 days continuous incubation) did not induce a direct anti BC effect in vitro (IC50&amp;gt;100 μM), but strongly affected stromal cell survival (IC50 8 μM). ZA treatment decreased TGFβ excretion by Hs27a cells dose dependently (ZA 10, 50, 100 or 500 μM for 48 hours) from 538±63 pg/ml (control) to 446±57 (ns), 285±98 (p&amp;lt;.01), 198±47 (p&amp;lt;.01) and 186±60 pg/ml (p&amp;lt;.001). In the co-culture, ZA (10, 50, 100 or 500 μM for 48 hours) also decreased TGFβ excretion dose dependently from 625±60 pg/ml (control) to 613±33 (ns), 535±83 (ns), 389±48 (p&amp;lt;.01) and 244±55 (p&amp;lt;.001) pg/ml. No TGFβ secretion by sole MCF-7 cells was detectable (&amp;lt;0±60 pg/ml). ZA had no effect on VEGF-A excretion by BC or stromal cells. On the CAM, co-cultures were sensitive to ZA treatment (73±8.27 vs 32±13.15 mm3 (p&amp;lt;.001)), whereas BC cells without stromal cells were resistant to ZA (tumor size: 22±14.35 vs 33±15 mm3 (ns)). In the co-cultures, tumor cell percentage evaluated by IHC tended to be smaller in the ZA treated group compared to control (31±15 vs 12±5 (ns)). Conclusion - Effects of therapeutic interventions on tumors by microenvironment modulation can be studied using the CAM assay. Our data suggest an indirect anti-BC effect of ZA in vivo via stromal cells in the BC microenvironment. This is accompanied by decreased TGFβ excretion, which may therefore function as biomarker for ZA treatment effect. Supported by Dutch Cancer Society grant RUG 2010-4739 Citation Format: Hilde H. Nienhuis, Marlous Arjaans, Hetty Timmer-Bosscha, Elisabeth G.E. de Vries, Carolina P. Schröder. Targeting a breast cancer xenograft by modulation of the microenvironment with zoledronic acid. [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 4950. doi:10.1158/1538-7445.AM2013-4950

Cancer continues to have a significant medical, economic, and psychological burden on patients worldwide. One strategy for reducing this impact involves the early detection of cancer biomarkers. Structural chromosomal aberrations called DNA palindromes have immense potential to be used as early markers in tumorigenesis. Palindromic sequences in the genome are structural genetic phenomena where a sequence of nucleotides is identical to its reverse compliment. Various independent studies have identified this distinctive pattern of genome instability in a wide range of cancers including breast, pancreatic, and ovarian cancers. We have shown previously that DNA palindromes underlie the amplification of ERBB2 in breast cancer. This suggests that DNA palindromes are the candidate breast cancer biomarker. A convenient and sensitive approach for identifying palindromes in breast tumor DNA could be a promising diagnostic test. Palindrome detection from tumor DNA in blood (liquid biopsy) could circumvent the need for invasive biopsies. Here, we develop an unbiased genome-wide technique for detecting DNA palindromes as a genomic target for breast cancer screening in the clinic. Using genome-wide analysis of palindrome formation (GAPF), we are able to enrich for DNA palindromes from samples of genomic DNA isolated from cells and tumor tissues. Following a denaturation step, DNA palindromes naturally form double-stranded DNA with itself because these sequences are physically tethered together and can form energetically favorable base pairs. Non-palindromic DNA remains single-stranded and subsequently removed by single-strand specific nucleases thereby dramatically improving the signal-to-noise ratio. This approach confers advantages over other methods because it simultaneously (1) amplifies target signal and (2) reduces background noise without employing sub-sampling techniques. We first used a cancer cell line Colo320DM containing a known palindromic junction and normal fibroblasts IMR90. By integrating GAPF with next generation sequencing (GAPF-Seq), we mapped sequenced reads to a reference genome and developed an algorithm to demarcate DNA palindromes throughout the genome. We are able to identify the known DNA palindrome in Colo320DM in a 10-fold dilution in IMR90 DNA. We then used GAPF-Seq to identify tumor-specific DNA palindromes in matched pairs of tumor and normal breast cancer tissue. From 100 ng of input DNA, we generated a profile of more than 300 tumor-specific DNA palindromes that were not found in the matched normal tissue. To optimize GAPF for use in cancer screening, we expect to detect DNA palindromes in the presence of 100-fold excess of non-palindromic DNA from as little as 10 ng of input DNA. We will then use matched plasma and buffy coat samples to generate whole-genome palindromic profiles from liquid biopsies. The data can also be used for tracing clonal evolution by comparing individual bins in the palindrome profiles of tumor tissue and plasma cell-free DNA from the same patient. Finally, we will optimize GAPF-Seq for minimal computational processing and data storage requirements. Due to the amplification of palindromes and reduction of background signal, GAPF-Seq may be able to identify tumor-specific breast cancer markers from as few as 1 million reads and 250 MB of data, which would be ideal for wide use in screening for breast tumors in the general population and monitoring disease outcomes with multiple blood samplings. We believe GAPF-Seq addresses a need to develop an unbiased, genome-wide, and rapid approach for breast cancer screening. Citation Format: Michael M. Murata, Lila Mouakkad, Dolores Di Vizio, Armando E. Giuliano, Hisashi Tanaka. DNA palindromes as novel genomic targets for unbiased, genome-wide, and rapid pan-cancer screening [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-24.

Breast cancer affects more than 1 million women per year worldwide. Through this study, we developed a nanoparticle-based drug delivery system to target breast cancer cells. Aspirin has been found to inhibit thromboembolic diseases with its tumor-preventing activity. As a consequence, it relieves disease symptoms and severity. Here, mesoporous silica nanoparticles (MNPs) have been used to deliver aspirin to the tumor location. MNP-based aspirin in folic acid (F)-conjugated polydopamine (MNP-Asp-PD-PG-F) vehicles are prepared for targeted breast cancer therapy. The vehicle hinges on MNP altered with polymer polyethylene glycol (PG), polydopamine (PD), and F. The delivery vehicle was studied for in vitro drug release, cytotoxicity, and breast cancer cell proliferation. F-conjugated drug delivery vehicles let MNPs achieve an elevated targeting efficacy, ideal for cancer therapy. It was also observed that compared to free aspirin, our drug delivery system (MNP-Asp-PD-PG-F) has a higher cytotoxic and antiproliferative effect on breast cancer cells. The drug delivery system can be proposed as a targeted breast cancer therapy that could be further focused on other targeted cancer therapies. Delivering aspirin by the PD-PG-F system on the tumor sites promises a therapeutic potential for breast cancer treatment.

Breast cancer is a leading cause of cancer-related mortality among women worldwide, characterized by its aggressive nature, propensity for metastasis, and resistance to standard treatment modalities. Traditional therapies, including surgery, chemotherapy, and radiation, often encounter significant limitations such as systemic toxicity and lack of specificity. This review aims to evaluate the recent advancements in phage-based nanomedicines as a novel approach for targeted breast cancer therapy, focusing on their mechanisms of action, therapeutic benefits, and the challenges faced in clinical implementation. A comprehensive literature review was conducted, analyzing studies that investigate the application of bacteriophages in cancer therapy, particularly in breast cancer. The review highlights the integration of nanotechnology with phage therapy, examining the potential for enhanced targeting and reduced side effects. Phage-based nanomedicines have shown promise in selectively targeting breast cancer cells while sparing healthy tissues, thereby improving therapeutic efficacy and safety profiles. The unique properties of bacteriophages, including their ability to be engineered for specific targeting and their natural ability to induce immune responses, present significant advantages over conventional treatments. The integration of phage therapy with nanotechnology represents a promising frontier in the fight against breast cancer. This review underscores the need for continued research to address existing challenges and to explore the full potential of phage-based nanomedicines in improving patient outcomes in breast cancer treatment.

Simple SummaryRheumatoid arthritis (RA) is one of the chronic autoimmune diseases that affects about 0.5 to 1.0% of the general population worldwide. The main symptom of RA is the destruction of the synovial joint, leading to a reduced quality of life and increased mortality. RA may be accompanied by several comorbidities, on which several studies have been conducted on the association between RA and breast cancer. However, the association between RA and breast cancer has shown different directions and has not been clearly established. In this study, we tried to determine whether RA had a causal effect on breast cancer using Mendelian randomization (MR) analysis, but causal evidence was not found. Therefore, additional studies are needed to determine whether RA patients are at high risk of breast cancer, based on large-scale cohorts to validate these results.Previous studies have been reported that the association between rheumatoid arthritis (RA) and breast cancer remains inconclusive. A two-sample Mendelian randomization (MR) analysis can reveal the potential causal association between exposure and outcome. A two-sample MR analysis using the penalized robust inverse variance weighted (PRIVW) method was performed to analyze the association between RA and breast cancer risk based on the summary statistics of six genome-wide association studies (GWAS) targeting RA in an East Asian population along with summary statistics of the BioBank Japan (BBJ), Breast Cancer Association Consortium (BCAC), and Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) targeting breast cancer. We found that the direction of the effect of RA on breast cancer varied among GWAS-summary data from BBJ, BCAC, and CIMBA. Significant horizontal pleiotropy based on a penalized robust MR-Egger regression was observed only for BBJ and CIMBA BRCA2 carriers. As the results of the two-sample MR analyses were inconsistent, the causal association between RA and breast cancer was inconclusive. The biological mechanisms explaining the relationship between RA and breast cancer were unclear in Asian as well as in Caucasians. Further studies using large-scale patient cohorts are required for the validation of these results.

BackgroundYWHAB (14-3-3 Beta) was found in the secretome of miR-526b and miR-655 overexpressed breast cancer (BRCA) cell lines. The potential of YWHAB as a therapeutic target or biomarker for BRCA is investigated here.MethodsAfter YWHAB was knocked down with siRNA, BRCA cell lines were used for in vitro assays (proliferation, migration, epithelial-to-mesenchymal transition). In silico analysis and in situ validation with BRCA plasma and biopsy tissues were used to test YWHAB’s biomarker potential.ResultsYWHAB RNA and protein expression are elevated in aggressive BRCA cell lines, and the knockdown of YWHAB inhibited cell migration, proliferation, and EMT in all subtypes of tumour cell lines. YWHAB expression is significantly higher in BRCA biopsy tissue and blood plasma compared to control tissues and benign plasmas. YWHAB is expressed in all hormonal subtypes of BRCA tumours and has shown increased expression in advanced tumour stages. Its high expression is linked to poor patient survival. YWHAB is a sensitivity tumour marker (AUC of 0.7340, p = 0.0012) but is not a promising blood biomarker. Nevertheless, combined with pri-miR-526b, YWHAB mRNA expression shows potential as a BRCA blood biomarker (AUC of 0.711, p = 0.032), which must be validated in a larger sample set.ConclusionWe elucidate the novel role of YWHAB as a therapeutic target in BRCA, given that its inhibition mitigated aggressive phenotypes across all tumour subtypes, including triple-negative breast cancer. Furthermore, YWHAB emerges as a potential tumour marker, exhibiting high expression in metastatic BRCA and correlating with poor patient survival; however, it is not a sensitive blood biomarker.

BackgroundCalmodulin 2 (CALM2) belongs to the highly conserved calcium-binding protein family, implicated in the pathogenesis of various malignant tumors. However, its involvement in breast cancer (BRCA) remains unclear. This study aimed to examine CALM2 expression in BRCA and its associations with prognosis, clinicopathological features, protein-protein interactions, and immune cell infiltration. Materials and MethodsOnline bioinformatics tools were employed to assess CALM2 expression and its clinical relevance in BRCA. Western blotting and immunohistochemistry were utilized to evaluate CALM2 expression in BRCA cell lines and tissues. Logistic regression was applied to analyze the relationship between CALM2 expression levels and clinicopathological parameters. Transwell assay was performed to validate the role of CALM2 in BRCA migration and invasion. ResultsCALM2 expression was significantly elevated in BRCA, with increased levels predicting poor overall survival (OS) and disease-free survival (DFS). Moreover, high CALM2 expression correlated with poorer DFS specifically in triple-negative breast cancer (TNBC). CALM2 expression in BRCA showed significant associations with lymph node metastasis, TP53 mutation status, and menopause status. Silencing CALM2 in BRCA cells demonstrated inhibition of cell migration and invasion in vitro. ConclusionsCALM2 is overexpressed in BRCA and its upregulation is significantly correlated with poor patient prognosis. Elevated CALM2 expression holds promise as a potential molecular marker for predicting poor survival and as a therapeutic target in BRCA.

6-phosphogluconate dehydrogenase (6PGD), a key enzyme of the oxidative pentose phosphate pathway, is involved in tumor growth and metabolism. Although high 6PGD activity has been shown to be associated with poor prognosis, its role and therapeutic value in breast cancer remain unknown. The levels and roles of 6PGD were analyzed in breast cancer cells and their normal counterparts. The underlying mechanisms of 6PGD's roles are also analyzed. We found that 6PGD is aberrantly activated in breast cancer as shown by its increased transcriptional and translational levels as well as enzyme activity in breast cancer tissues and cell lines compared to normal counterparts. Although similar degree of enzyme activity inhibition was achieved in both breast cancer and normal breast cells, 6PGD inhibition by siRNA-mediated knockdown or pharmacological inhibitor physcion is more effective in inhibiting growth and survival in breast cancer than normal breast cells. Moreover, inhibiting 6PGD significantly sensitizes breast cancer response to chemotherapeutic agents in in vitro cell culture system and in vivo xenograft breast cancer model. We further show that 6PGD inhibition activates AMPK and its downstream substrate ACC1, leading to reduction of ACC1 activity and lipid biosynthesis. AMPK depletion significantly reverses the inhibitory effects of physcion in breast cancer cells, confirming that 6PGD inhibition targets breast cancer cell via AMPK activation. Our work provides experimental evidence on the association of 6PGD with poor prognosis in breast cancer and suggests that 6PGD inhibition may represent a potential therapeutic strategy to augment chemotherapy efficacy in breast cancer.

Targeting breast cancer using phytoconstituents: Nanomedicine-based drug delivery

Chapter 16 - Targeting breast cancer

To present the changes in 5-year relative survival of patients with breast and colorectal cancer in a northern Italian province in the 2000-2012 period. Changes are presented in relation to stage, period, and age in the screening population. A total of 5073 breast cancer and 4093 colorectal cancer cases were registered. Breast cancer was divided into 5 age groups: <45, 45-49, 50-69, 70-74 (the target screening population is ages 45-74), and >74 years. Colorectal cancers were classified into 3 age groups: <50, 50-69 (screening target population), and >69 years. Survival was analysed by stage, period, and screening program. Five-year breast cancer relative survival was 92.9% overall, with 100%, 91.9%, 78.8%, 34.2%, and 76.4% for stages I, II, III, IV, and unknown, respectively. Survival increased from 89.7%, 91.8%, and 93.4%, respectively, in the periods 2000-2005, 2006-2009, and 2010-2012. Breast cancer survival was stable in early stages (although already high); a slight improvement was seen for stages III and IV. Five-year colorectal cancer relative survival was 63.6% overall, with 93.3%, 78.6%, 69.8%, 13.2%, and 55.8% for stages I, II, III, IV, and unknown, respectively. No improvement in survival was seen in the periods analysed, although the introduction of screening in 2005 did lead to a sharp decrease in incidence and mortality. For breast cancer, extending the screening target age led to an improvement in survival; for colorectal cancer, the best impact was on incidence and mortality rate.

Breast cancer is the leading cause of death among women worldwide. Despite the recent treatment options like surgery, chemotherapy etc. the lethality of breast cancer is alarming. Natural compounds are considered a better treatment option against breast carcinoma because of their lower side effects and specificity in targeting important proteins involved in the aberrant activation of pathways in breast cancer. A recently discovered compound called Juglanthraquinone C, which is found in the bark of the Juglans mandshurica Maxim (Juglandaceae) tree has shown promising cytotoxicity in hepatocellular carcinoma. However, not much data is available on the molecular mechanisms followed by this compound. Therefore, we aimed to investigate the molecular mechanism followed by Juglanthraquinone C against breast cancer. We used the network pharmacology technique to analyse the mechanism of action ofJuglanthraquinone Cin breast cancer and validated our study by applying various computational tools such as UALCAN, cBioportal, TIMER, docking and simulation. The results showed the compound and breast cancer target network shared 31 common targets. Moreover, we observed thatJuglanthraquinone C targets multiple deregulated genes in breast cancer such as TP53, TGIF1, IGF1R, SMAD3, JUN, CDC42, HBEGF, FOS and signaling pathways such as PI3K-Akt pathway, TGF-β signaling pathway, MAPK pathway and HIPPO signaling pathway. A docking examination revealed that the investigated drug had a high affinity for the primary target TGIF1 protein. A stable protein–ligand combination was generated by the best hit molecule, according to molecular dynamics modeling. The main aim of this study was to examine Juglanthraquinone C's significance as a prospective breast cancer treatment and to better understand the molecular mechanism this substance uses in breast cancer since there is a need to discover new therapeutics to decrease the load on current therapeutics which also are currently ineffective due to several side effects and development of drug resistance.

Breast cancer is the most common cancer and the second leading cause of cancer-related deaths among women worldwide. Although patients are often diagnosed in the early and curable stages, the treatment of metastatic breast cancer remains a major clinical challenge. The combination of chemotherapy with new targeting agents, such as bevacizumab, is helpful in improving patient survival; however, novel treatment strategies are required to improve clinical outcomes. The insulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase cell surface receptor which is involved in the regulation of cell growth and metabolism. Previous studies have shown that activation of the IGF-IR signaling pathway promotes proliferation, survival, and metastasis of breast cancer cells. Additionally, overexpression of IGF-IR is associated with breast cancer cell resistance to anticancer therapies. Recently, IGF-IR has been introduced as a marker of stemness in breast cancer cells and there is also accumulating evidence that IGF-IR contributes to the establishment and maintenance of breast cancer epithelial-mesenchymal transition (EMT). Therefore, pharmacological or molecular targeting of IGF-IR could be a promising strategy, in the treatment of patients with breast cancer, particularly in order to circumvent the therapeutic resistance and targeting breast cancer stem/progenitors. Currently, many strategies have been developed for targeting IGF-IR, some have entered clinical trials and some are in preclinical stages for breast cancer therapy. In this review, we will first discuss on the biology of IGF-IR in an attempt to find the role of this receptor in breast cancer and then discuss about therapeutic strategies to target this receptor.

ObjectiveHuman endogenous retroviruses (HERVs) make up 8% of the human genome. HERVs are biologically active elements related to multiple diseases. HERV-K, a subfamily of HERVs, has been associated with certain types of cancer and suggested as an immunologic target in some tumors. The expression levels of HERV-K in breast cancer (BCa) have been studied as biomarkers and immunologic therapeutic targets. However, HERV-K has multiple copies in the human genome, and few studies determined the transcriptional profile of HERV-K copies across the human genome for BCa.MethodsNinety-one HERV-K indexes with entire proviral sequences were used as the reference database. Nine raw sequencing datasets with 243 BCa and 137 control samples were mapped to this database by Salmon software. The differential proviral expression across several groups was analyzed by DESeq2 software.ResultsFirst, the clustering of each dataset demonstrated that these 91 HERV-K proviruses could well cluster the BCa and control samples when the normal controls were normal cells or healthy donor tissues. Second, several common HERV-K proviruses that are closely related with BCa risk were significantly differentially expressed (padj < 0.05 and absolute log2FC > 1.5) in the tissues and cell lines. Additionally, almost all the HERV-K proviruses had higher expression in BCa tissue than in healthy donor tissue. Notably, we first found the expression of 17p13.1 provirus that located with TP53 should regulate TP53 expression in ER+ and HER2+ BCa.ConclusionThe expression profiling of these 91 HERV-K proviruses can be used as biomarkers to distinguish individuals with BCa and healthy controls. Some proviruses, especially 17p13.1, were strongly associated with BCa risk. The results suggest that HERV-K expression profiles may be appropriate biomarkers and targets for BCa.