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TP53 (p53) is mutated in 80–90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines (atorvastatin, p p

Mutant p53 is one of the most attractive targets for new anti-cancer drugs. Although traditionally regarded as difficult to drug, several new strategies have recently become available for targeting the mutant protein. One of the most promising of these involves the use of low molecular weight compounds that promote refolding and reactivation of mutant p53 to its wild-type form. Several such reactivating drugs are currently undergoing evaluation in clinical trials, including eprenetapopt (APR-246), COTI-2, arsenic trioxide and PC14586. Of these, the most clinically advanced for targeting mutant p53 is eprenetapopt which has completed phase I, II and III clinical trials, the latter in patients with mutant TP53 myelodysplastic syndrome. Although no data on clinical efficacy are currently available for eprenetapopt, preliminary results suggest that the drug is relatively well tolerated. Other strategies for targeting mutant p53 that have progressed to clinical trials involve the use of drugs promoting degradation of the mutant protein and exploiting the mutant protein for the development of anti-cancer vaccines. With all of these ongoing trials, we should soon know if targeting mutant p53 can be used for cancer treatment. If any of these trials show clinical efficacy, it may be a transformative development for the treatment of patients with cancer since mutant p53 is so prevalent in this disease.

Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as “undruggable” or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC50 values varying form 0.3 μM to >10 μM. Consistent with its ability to decrease cell growth, MYCMI-6 was found to induce apoptosis in two cell lines in which growth was inhibited but not in two cell lines that were resistant to growth inhibition. Across all breast cancers, MYC was found to be amplified in 15.3% of cases in the TCGA database and 26% in the METABRIC database. Following classification of the breast cancers by their molecular subtypes, MYC was most frequently amplified and exhibited highest expression at both mRNA and protein level in the basal subtype. Conclusions Based on these findings, we conclude that for patients with breast cancer, anti-MYC therapy is likely to be most efficacious in patients with the basal subtype.

Background MYC is one of the most frequently altered driver genes in triple-negative breast cancer (TNBC). The aim of this study was to evaluate targeting MYC for the treatment of TNBC. Methods The anti-proliferative and apoptosis-inducing effects of the recently discovered MYC inhibitor, MYCi975 were investigated in a panel of 14 breast cancer cell lines representing the main molecular forms of breast cancer. Results IC50 values for growth inhibition by MYCi975 varied from 2.49 to 7.73 µM. Response was inversely related to endogenous MYC levels as measured by western blotting (p = 0.047, r = − 0.5385) or ELISA (p = 0.001, r = − 0.767), i.e., response to MYCi975 decreased as endogenous MYC levels increased. MYCi975 also induced variable levels of apoptosis across the panel of cell lines, ranging from no detectable induction to 80% induction. Inhibition of proliferation and induction of apoptosis were greater in TNBC than in non-TNBC cell lines (p = 0.041 and p = 0.001, respectively). Finally, combined treatment with MYCi975 and either paclitaxel or doxorubicin resulted in enhanced cell growth inhibition. Discussion Our findings open the possibility of targeting MYC for the treatment of TNBC. Based on our results, we suggest that trials use a combination of MYCi975 and either docetaxel or doxorubicin and include MYC as a putative therapy predictive biomarker.

TP53 (p53) is mutated in 80-90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines (atorvastatin, p 0.01; simvastatin p 0.05) indicating greater sensitivity. Furthermore, cell lines containing mutant p53 were more responsive to both statins than cell lines expressing wild-type p53, suggesting that the mutational status of p53 is a potential predictive biomarker for statin response. In addition to inhibiting proliferation, simvastatin was also found to promote cell cycle arrest and induce apoptosis. Using an apoptosis array capable of detecting 43 apoptosis-associated proteins, a novel protein shown to be upregulated by simvastatin was the IGF-signalling modulator, IGBP4, a finding we confirmed by Western blotting. Finally, we found synergistic growth inhibition between simvastatin and the IGF-1R inhibitor, OSI-906 as well as between simvastatin and doxorubicin or docetaxel. Our work suggests repurposing of statins for clinical trials in patients with TNBC. Based on our findings, we suggest that these trials investigate statins in combination with either doxorubicin or docetaxel and include p53 mutational status as a potential predictive biomarker.

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and β-glycerophosphate (βG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to βG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with βG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples., The mineralization of MDA-MB-231 breast cells was investigated using Raman micro-spectroscopy. Mineralization was induced by two osteogenic agents: inorganic phosphate (Pi) and β-Glycerophosphate (βG). The results show that the uptake of Pi allows a faster mineralization and the uptake of βG indicated the presence of a precursor phase during the hydroxyapatite crystal formation.

Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as "undruggable" or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC

Dysfunction of the TP53 (p53) gene occurs in most if not all human malignancies. Two principal mechanisms are responsible for this dysfunction; mutation and downregulation of wild-type p53 mediated by MDM2/MDM4. Because of its almost universal inactivation in malignancy, p53 is a highly attractive target for the development of new anticancer drugs. Although multiple strategies have been investigated for targeting dysfunctional p53 for cancer treatment, only 2 of these have so far yielded compounds for testing in clinical trials. These strategies include the identification of compounds for reactivating the mutant form of p53 back to its wild-type form and compounds for inhibiting the interaction between wild-type p53 and MDM2/MDM4. Currently, multiple p53-MDM2/MDM4 antagonists are undergoing clinical trials, the most advanced being idasanutlin which is currently undergoing testing in a phase III clinical trial in patients with relapsed or refractory acute myeloid leukemia. Two mutant p53-reactivating compounds have progressed to clinical trials, i.e., APR-246 and COTI-2. Although promising data has emerged from the testing of both MDM2/MDM4 inhibitors and mutant p53 reactivating compounds in preclinical models, it is still unclear if these agents have clinical efficacy. However, should any of the compounds currently being evaluated in clinical trials be shown to have efficacy, it is likely to usher in a new era in cancer treatment, especially as p53 dysfunction is so prevalent in human cancers.

Background: Despite many advances in recent years, therapeutic options for triple-negative breast cancer (TNBC) remain limited by a lack of defined molecular targets. Many studies have found that cholesterol-lowering statins possess significant anti-tumor effects, including against TNBC. Statins influence several key signaling pathways and have recently been shown to degrade mutant p53, a common oncogenic driver. We aim to further evaluate the efficacy of statins in the treatment of TNBC and examine links with the p53 pathway. Methods: The anti-proliferative effect of two different statins, atorvastatin and simvastatin was tested on a panel of 15 breast cancer cell lines (8 TNBC & 7 non-TNBC). Viability was measured using the MTT assay at 5 days incubation and colony formation assays at 10 days. Induction of apoptosis was analyzed by flow cytometry following annexin V staining. Western blotting was used to assess levels of mutant-p53. Results: Both atorvastatin and simvastatin significantly reduced proliferation in a panel of breast cancer cell lines (IC50 values: atorvastatin 0.3 - 49.1 µM, simvastatin 0.2 - 40.8 µM). Simvastatin was selected for further study due to its significantly lower mean IC50 values (11.85 v 17.69 µM, p Addition of the cholesterol-precursor compound MVA negated the anti-proliferative effect of statins, suggesting the crucial nature of this pathway in TNBC cell survival. Combination treatment of simvastatin and the chemotherapeutic agents, doxorubicin or docetaxel showed synergistic growth inhibition. Finally, analysis of apoptosis by annexin-V staining found significant increases in statin treated cells (p Conclusions: Our results suggest that statins may be a new treatment for patients with breast cancer especially those with TNBC. Preliminary results also suggest the presence of mutant-p53 may be both a marker of statin response and a mediator by which the statins act. Future work will aim to further clarify the precise signaling pathways affected by these drugs and the mechanism by which they induce cell death. Citation Format: Shane O'Grady, John Crown, Michael J. Duffy. Anti-tumor effects of statins in triple-negative breast cancer: Apoptosis, chemosensitization and degradation of mutant-p53 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1775.

Microcalcifications are vital mammographic indicators contributing to the early detection of up to 50% of non-palpable tumours and may also be valuable as prognostic markers. However, the precise mechanism by which they form remains incompletely understood. Following development of an in vitro model using human breast cancer cells lines cultured with a combination of mineralisation-promoting reagents, analysis of calcium deposition, alkaline phosphatase (ALP) activity and changes in expression of key genes was used to monitor the calcification process. Two cell lines were identified as successfully mineralising in vitro, MDA-MB-231 and SKBR3. Mineralising cell lines displayed higher levels of ALP activity that was further increased by addition of mineralisation promoting media. qPCR analysis revealed changes in expression of both pro- (RUNX2) and anti- (MGP, ENPP1) mineralisation genes. Mineralisation was suppressed by chelation of intracellular Ca2+ and inhibition of TRPM7, demonstrating a functional role for the channel in formation of microcalcifications. Increased Mg2+ was also found to effectively reduce calcium deposition. These results expand the number of human breast cancer cell lines with a demonstrated in vitro mineralisation capability, provide further evidence for the role of an active, cellular process of microcalcification formation and demonstrate for the first time a role for TRPM7 mediated Ca2+ transport.

The implementation of mammographic screening programmes in many countries has been linked to a marked increase in early detection and improved prognosis for breast cancer patients. Breast tumours can be detected by assessing several features in mammographic images but one of the most common are the presence of small deposits of calcium known as microcalcifications, which in many cases may be the only detectable sign of a breast tumour. In addition to their efficacy in the detection of breast cancer, the presence of microcalcifications within a breast tumour may also convey useful prognostic information. Breast tumours with associated calcifications display an increased rate of HER2 overexpression as well as decreased survival, increased risk of recurrence, high tumour grade and increased likelihood of spread to the lymph nodes. Clearly, the presence of microcalcifications in a tumour is a clinically significant finding, suggesting that a detailed understanding of their formation may improve our knowledge of the early stages of breast tumourigenesis, yet there are no reports which attempt to bring together recent basic science research findings and current knowledge of the clinical significance of microcalcifications. This review will summarise the most current understanding of the formation of calcifications within breast tissue and explore their associated clinical features and prognostic value.

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer.

Background: Microcalcifications resulting from calcium deposition in the mammary gland play a central role in the early detection of breast cancer [1]. However, the relationship between their occurrence in the breast and cancer progression remains poorly understood. Our approach is to use vibrational spectroscopy and imaging, which is non-invasive, non-destructive, label-free and chemically specific, to assess the composition and distribution of the deposits in an in vitro cancer cell model of mineralisation [2]. In parallel we will utilise the same methods to measure the biochemical composition of microcalcifications found in breast biopsies from different grades of cancer. The ultimate aim of the study is to link the changes identified during the in vitro mineralisation process with the different stages of breast cancer. Vibrational spectroscopic methods can provide incredibly detailed biomolecular fingerprints enabling us to elucidate both the compositional changes with advancing pathology and the spatial distribution of those changes within the calcification and the surrounding tissue. Methods: The breast cancer cell line MDA-MB-231 has the ability to produce mineralisation. This mineralisation was assessed over a 14-day period in the presence of different osteogenic cocktails: one composed of ascorbic acid, β-glycerophosphate (βG) and dexamethasone (Dex), and another one composed of inorganic phosphate (Pi). Fixed cells were analysed using Raman spectroscopy and micro-FTIR imaging at different time points (3, 7, 11 and 14 days). Tissue sections from patients with microcalcifications identified in histopathology will be sectioned to 3 mm and imaged with infrared (Agilent 670 FTIRinterferometer and Focal Plane Array imaging microscope) and Raman (Renishaw inVia) microspectrometers. Results: We observed distinct and specific phosphate peak (PO43-) at 960 and 1020 cm-1 in Raman and FTIR spectra, respectively, corresponding to hydroxyapatite crystal and indicating the presence of microcalcification formation. Treatment with Pi induced a faster mineralisation (day 3) compared to cells treated with βG (day 11) and different spectral profiles during this development phase. In addition, there are changes in both the relative DNA and protein concentrations in the cells following 11 days exposure to the osteogenic agents. It has been shown that the level of carbonate substitution in the calcium hydroxyapatite crystal correlates directly with the pathology of the tissue surrounding the microcalcification. Here we compare the mineral composition found ex vivo versus the in vitro model. Conclusion: It could be possible to link the progressive biophysical changes associated with mineralisation to distinct stages of breast cancer pathology based on protein, lipid and carbonated apatite contents of the mineralised cells. Support:This work was conducted as part of the Marie Curie Innovative Training Network Mid-TECH [H2020-MSCA-ITN-2014-642661] References: [1] R. Baker, KD. Rogers, N. Shepherd and N. Stone. British Journal of Cancer. 103, 1034-1039 (2010) [2] RF. Cox, A. Hernandez-Santana, S. Ramdass, G. McMahon, JH. Harmey and MP. Morgan. British Journal of Cancer. 106, 525–537 (2012) Citation Format: Bouzy P, O'Grady S, Palombo F, Morgan MP, Stone N. Exploring the relationship between an in vitro model of breast cancer cell mineralisation and the cancer grade specific composition of ex vivo microcalcifications [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 P6-01-03.

Antimicrobial Peptides (AMPs) have unique anticancer properties, but their clinical application is currently limited by an inadequate margin of safety. A prodrug strategy associated with a combination therapy approach could address this limitation by increasing their therapeutic index and their efficacy. Accordingly, the first targeted anticancer polymeric prodrug candidates of AMPs, intended for combination therapy with another polymeric prodrug of an approved antineoplastic agent (doxorubicin), were synthesized as either a PEG-based dual-release prodrug or two individual pegylated prodrugs. The latter are based on a cathepsin B-labile peptide linker and an acid-sensitive acyl hydrazone bond for the AMP and doxorubicin prodrugs, respectively. Anticancer activities and toxicity differentials achieved with the free peptide and its polymer conjugates against ovarian, cancer and non-malignant, cells, indicate that protease-dependent reversible pegylation could be implemented to increase the therapeutic indices of AMPs in cancer therapy. The results obtained also show that this approach can be developed if the releasable PEG linker can be optimised to conciliate the attributes and restrictions of pegylation against proteases. In addition, combination of the polymeric prodrugs of the AMP and of doxorubicin provides additive antitumor effects which could be exploited to enhance the efficacy of the AMP candidate.

Liver cancer is global health problem, claiming hundreds of thousands of deaths every year. Despite significant efforts by doctors, scientists and the pharmaceutical industry, treatment options for hepatocellular carcinoma (HCC) remain poor, with 5-year survival rates of approximately 15%. The paucity of viable clinical treatments available for HCC patients has prompted many to look outside the boundaries of traditional cytotoxic chemotherapeutic and consider targeting the epigenetic make up of malignant hepatocytes. Most commonly, this approach is aimed at altering the activity of histone deacetylases, a family of 18 proteins that play an important role in gene regulation, along with DNA methylation. The role of central epigenetic mechanisms in cancer treatment was originally proposed to result from a normalization of aberrant gene regulation within tumors but is now understood to be far more wide reaching, with significant effects demonstrated against a variety of cellular processes, including metabolism, migration, proliferation, and endoplasmic reticulum stress. The advent of powerful tools in the form of epigenetic agents that have the potential to alter the course of HCC has been highlighted. Nonetheless, this rough diamond needs further refinement and more clarity to truly access their potential benefit. Increases in HCC cases hand in hand with limited treatment options and persistently low survival rates make epigenetic agents a glimmer of hope in the fight against liver cancer.

The presence of microcalcifications on mammographic images represents a highly valuable tool in the early detection of breast cancer, often allowing radiographers to diagnose mammary tumours at an early, in-situ stage. The presence of microcalcifications has also been linked to a number of unfavourable prognostic factors including decreased survival and an increased probability of relapse. Despite their long history of use in the clinic, the precise mechanisms by which microcalcifications are formed remains poorly understood. Pathological soft-tissue calcification is often considered the result of an imbalance between pro- and anti-calcifying factors. However, the possible impact of such an imbalance has not been studied in the context of breast calcification. We have established an in-vitro model of microcalcification formation using the triple-negative adenocarcinoma cell line MDA-MB-231. When cultured in the presence of the osteogenesis-promoting reagents β-glycerophosphate, ascorbic acid and dexamethasone, calcified deposits begin to form by Day 14, as verified by Alizarin Red staining and the quantitative o-cresolphthalein assay. Following the establishment of our model, we set out to identify the underlying molecular triggers initiating the calcification process, in particular the role of micro-environmental factors. Altered magnesium homeostasis has been suggested as an important mediator of tissue calcification. We found that a slight increase in magnesium concentration almost completely blocked calcium deposition in our model. Previous studies in other tissues have shown this protective effect to be dependent on the cation channel TRPM7 which is increased in breast cancer patients with calcifications compared to those without. Unexpectedly, inhibition of the TRPM7 channel by two separate compounds (2-APB and NS-8593) not only failed to reverse the inhibition of microcalcification formation by exogenous magnesium, but actually further decreased calcium deposition by Day 28, suggesting that calcium influx via the TRPM7 channel may be promoting development of mammary calcifications. Finally, we investigated the effect of several tumour associated cytokines on the rate of calcium deposition and found that IL-1β and TNF-α blocked mineralisation whilst IL-6 and BMP2 lead to an increase. Interestingly, co-administration of IL-6 alongside a soluble form of its receptor (sIL-6R) was observed to promote mineralisation even in the absence of dexamethasone, which had previously been essential to the formation of calcifications in our model. To date, our model has yielded a number of important insights into the formation of calcifications in breast cell lines, many of which recapitulate observations from patient studies. It is hoped that this work will contribute to our understanding of the origin of these pre-invasive diagnostic clues. Citation Format: Shane O'Grady, Maria P. Morgan. Tumour micro-environmental factors and the development of mammary calcifications: Recent findings from an in-vitro model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2017-799