BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, lacking effective targeted therapies, and whose underlying mechanisms are still unclear. The gene coding for Gametogenetin-binding protein (GGNBP2), also known as Zinc Finger Protein 403 (ZNF403), is located on chromosome 17q12-q23, a region known as a breast cancer susceptibility locus. We have previously reported that GGNBP2 functions as a tumor suppressor in estrogen receptor-positive breast cancer. The aim of this study was to evaluate the role and mechanisms of GGNBP2 in TNBC.MethodsThe effect of GGNBP2 on TNBC aggressiveness was investigated both in vitro and in vivo. The protein and mRNA expression levels were analyzed by western blotting and reverse transcription quantitative polymerase chain reaction, respectively. Fluorescence-activated cell sorting analysis was used to evaluate the cell cycle distribution and cell apoptosis. Immunohistochemistry was used to determine the expression of GGNBP2 in breast cancer tissues.ResultsWe find that GGNBP2 expression decreases in TNBC tissues and is associated with the outcome of breast cancer patients. Furthermore, experimental overexpression of GGNBP2 in MDA-MB-231 and Cal51 cells suppresses cell proliferation, migration and invasion, reduces the cancer stem cell subpopulation, and promotes cell apoptosis in vitro as well as inhibits tumor growth in vivo. In these cell models, overexpression of GGNBP2 decreases the activation of IL-6/STAT3 signaling.ConclusionOur data demonstrate that GGNBP2 suppresses cancer aggressiveness by inhibition of IL-6/STAT3 activation in TNBC.

STK11 adnexal tumour is a recently described female genital tract tumour, usually identified in a paratubal location, often associated with Peutz-Jeghers syndrome (PJS) and with STK11 gene alterations identified in most of the cases. Morphologically, this tumour is composed of cells arranged in a variety of patterns, including cords, trabeculae, tubules and cystic and acinar structures. The cells are only moderately pleomorphic and mitotic activity is variable. As tumour cells express epithelial, sex cord stromal and mesothelial markers, STK11 adnexal tumour may be of sex cord stromal, epithelial or mesothelial origin; a Wolffian origin has also been suggested. We report the ultrastructural features of two STK11 adnexal tumours and compare their ultrastructural features with those of other sex cord stromal tumours, a granulosa cell tumour cell line, as well as the known ultrastructural features of epithelial, mesothelial and Wolffian cells. On ultrastructural examination, two STK11 adnexal tumours showed an admixture of elongated cells with regular elongated nuclei and polygonal cells with nuclei showing markedly irregular outlines and prominent nucleoli. Extracellular collagen fibres were identified. These are common ultrastructural features of sex cord stromal tumours, principally sex cord tumour with annular tubules; no ultrastructural features of epithelial, mesothelial or Wolffian cells were found. These findings in conjunction with the shared clinical and genetic association with PJS and shared molecular changes in STK11 gene suggest that STK11 adnexal tumour represents a poorly differentiated sex cord tumour.

Background/Objectives: Serous ovarian carcinoma (SOC) is the most common subtype of epithelial ovarian cancer, with high-grade (HGSOC) and low-grade (LGSOC) subtypes presenting distinct clinical behaviours. This study aimed to evaluate histopathologic features in SOC, correlating these with prognostic outcomes, and explore the potential clinical implications. Methods: We analysed 51 SOC cases for lymphovascular space invasion (LVSI), tumour border configuration (TBC), microvessel density (MVD), tumour budding (TB), the tumour-stroma ratio (TSR), the stromal type, tumour-infiltrating lymphocytes (TILs), and tertiary lymphoid structures (TLSs). A validation cohort of 54 SOC cases from The Cancer Genome Atlas (TCGA) was used for comparison. Results: In the discovery set, significant predictors of aggressive behaviour included LVSI, high MVD, high TB, and low TILs. These findings were validated in the validation set where the absence of TLSs, lower peritumoural TILs, immature stromal type, and low TSR were associated with worse survival outcomes. The stromal type was identified as an independent prognostic predictor in SOC across both datasets. Inter-observer variability analysis demonstrated substantial to almost perfect agreement for these features, ensuring the reproducibility of the findings. Conclusions: The histopathological evaluation of immune and stromal features, such as TILs, TLSs, TB, TSR, and stromal type, provides critical prognostic information for SOC. Incorporating these markers into routine pathological assessments could enhance risk stratification and guide treatment, offering practical utility, particularly in low-resource settings when molecular testing is not feasible.

When ectopically overexpressed, anticancer genes, such as TRAIL, PAR4 and ORCTL3, specifically destroy tumour cells without harming untransformed cells. Anticancer genes can not only serve as powerful tumour specific therapy tools but studying their mode of action can reveal mechanisms underlying the neoplastic transformation, sustenance and spread. Anticancer gene discovery is normally accidental. Here we describe a systematic, gain of function, forward genetic screen in mammalian cells to isolate novel anticancer genes of human origin. Continuing with over 30,000 transcripts from our previous study, 377 cell death inducing genes were subjected to screening. FBLN5 was chosen, as a proof of principle, for mechanistic gene expression profiling, comparison pathways analyses and functional studies. Sixteen novel anticancer genes were isolated; these included non-coding RNAs, protein-coding genes and novel transcripts, such as ZNF436-AS1, SMLR1, TMEFF2, LINC01529, HYAL2, NEIL2, FBLN5, YPEL4 and PHKA2-processed transcript. FBLN5 selectively caused inhibition of MYC in COS-7 (transformed) cells but not in CV-1 (normal) cells. MYC was identified as synthetic lethality partner of FBLN5 where MYC transformed CV-1 cells experienced cell death upon FBLN5 transfection, whereas FBLN5 lost cell death induction in MCF-7 cells upon MYC knockdown. Sixteen novel anticancer genes are present in human genome including FBLN5. MYC is a synthetic lethality partner of FBLN5.

Supplementary Figure Legends 1-2 from Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Supplementary Table 5 from Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Drug resistance is a major obstacle to the successful treatment of cancer. The role of the miR-106b-25 cluster in drug resistance of haematologic malignancies has not yet been elucidated. Here, we show that the miR-106b-25 cluster mediates resistance to therapeutic agents with structural and mechanistic dissimilarity in vitro and in vivo. RNA sequencing data revealed that overexpression of the miR-106b-25 cluster or its individual miRNAs resulted in downregulation of multiple key regulators of apoptotic pathways. Luciferase reporter assay identified TP73 as a direct target of miR-93 and miR-106b, BAK1 as a direct target of miR-25 and CASP7 as a direct target of all three miRNAs. We also showed that inhibitors of the miR-106b-25 cluster and BCL-2 exert synergistic effects on apoptosis induction in primary myeloid leukaemic cells. Thus, the members of the miR-106b-25 cluster may jointly contribute to myeloid leukaemia drug resistance by inactivating multiple apoptotic genes. Targeting this cluster could be a promising combination strategy in patients resistant to therapeutic agents that induce apoptosis.

Supplementary Table 3 from Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Resistance to cancer therapeutics represents a leading cause of mortality and is particularly important in cancers, such as triple negative breast cancer, for which no targeted therapy is available, as these are only treated with traditional chemotherapeutics. Cancer, as well as bacterial, drug resistance can be intrinsic, acquired or adaptive. Adaptive cancer drug resistance is gaining attention as a mechanism for the generation of long-term drug resistance as is the case with bacterial antibiotic resistance. We have used a cellular model of triple negative breast cancer (CAL51) and its drug resistance derivative (CALDOX) to gain insight into genome-wide expression changes associated with long-term doxorubicin (a widely used anthracycline for cancer treatment) resistance and doxorubicin-induced stress. Previous work indicates that both naïve and resistance cells have a functional p53-p21 axis controlling cell cycle at G1, although this is not a driver for drug resistance, but down-regulation of TOP2A (topoisomerase IIα). As expected, CALDOX cells have a signature characterized, in addition to down-regulation of TOP2A, by genes and pathways associated with drug resistance, metastasis and stemness. Both CAL51 and CALDOX stress signatures share 12 common genes (TRIM22, FAS, SPATA18, SULF2, CDKN1A, GDF15, MYO6, CXCL5, CROT, EPPK1, ZMAT3 and CD44), with roles in the above-mentioned pathways, indicating that these cells have similar functional responses to doxorubicin relaying on the p53 control of apoptosis. Eight genes are shared by both drug stress signatures (in CAL51 and CALDOX cells) and CALDOX resistant cells (FAS, SULF2, CDKN1A, CXCL5, CD44, SPATA18, TRIM22 and CROT), many of them targets of p53. This corroborates experimental data indicating that CALDOX cells, even in the absence of drug, have activated, at least partially, the p53-p21 axis and DNA damage response. Although this eight-gene signature might be an indicator of adaptive resistance, as this transient phenomenon due to short-term stress may not revert to its original state upon withdrawal of the stressor, previous experimental data indicates that the p53-p21 axis is not responsible for doxorubicin resistance. Importantly, TOP2A is not responsive to doxorubicin treatment and thus absent in both drug stress signatures. This indicates that during the generation of doxorubicin resistance, cells acquire genetic changes likely to be random, leading to down regulation of TOP2A, but selected during the generation of cells due to the presence of drug in the culture medium. This poses a considerable constraint for the development of strategies aimed at avoiding the emergence of drug resistance in the clinic.

Supplementary Figure 1 from Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Supplementary Figure 2 from Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Supplementary Table 4 from Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Traditional Chinese medicine (TCM) has used herbal remedies for more than 2,000 years. The use of complimentary therapies has increased dramatically during the last years, especially in the West, and the incorporation and modernization of TCM in current medical practice is gaining momentum. We reflect on the main bottlenecks in the modernization of arcane Chinese herbal medicine: lack of standardization, safety concerns and poor quality of clinical trials, as well as the ways these are being overcome. Progress in these areas will facilitate the implementation of an efficacy approach, in which only successful clinical trials lead to the molecular characterization of active compounds and their mechanism of action. Traditional pharmacological methodologies will produce novel leads and drugs, and we describe TCM successes such as the discovery of artemisinin as well as many others still in the pipeline. Neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, cancer and cardiovascular disease are the main cause of mortality in the Western world and, with an increasing old population in South East Asia, this trend will also increase in the Far East. TCM has been used for long time for treating these diseases in China and other East Asian countries. However, the holistic nature of TCM requires a paradigm shift. By changing our way of thinking, from "one-target, one-drug" to "network-target, multiple-component-therapeutics," network pharmacology, together with other system biology methodologies, will pave the way toward TCM modernization.

Triple-negative metaplastic breast carcinoma (MBC) poses a significant treatment challenge due to lack of targeted therapies and chemotherapy resistance. We isolated a novel MBC cell line, BAS, which showed a molecular and phenotypic profile different from the only other metaplastic cell model, HS578T cells. To gain insight behind chemotherapeutic resistance, we generated doxorubicin (HS-DOX, BAS-DOX) and paclitaxel (HS-TX, BAS-TX) resistant derivatives of both cell lines. Drug sensitivity assays indicated a truly multidrug resistant (MDR) phenotype. Both BAS-DOX and BAS-TX showed up-regulation of FOXC1 and its experimental down-regulation re-sensitized cells to doxorubicin and paclitaxel. Experimental modulation of FOXC1 expression in MCF-7 and MDA-MB-231 cells corroborated its role in MDR. Genome-wide expression analyses identified gene expression signatures characterized by up-regulation of TGFB2, which encodes cytokine TGF-β2, in both BAS-DOX and BAS-TX cells. Pharmacological inhibition of the TGF-β pathway with galunisertib led to down-regulation of FOXC1 and increase in drug sensitivity in both BAS-DOX and BAS-TX cells. MicroRNA (miR) expression analyses identified high endogenous miR-495-3p levels in BAS cells that were downregulated in both BAS MDR cells. Transient expression of miR-495-3p mimic in BAS-DOX and BAS-TX cells caused downregulation of TGFB2 and FOXC1 and re-sensitized cells to doxorubicin and paclitaxel, whereas miR-495-3p inhibition in BAS cells led to increase in resistance to both drugs and up-regulation of TGFB2 and FOXC1. Together, these data suggest interplay between miR-495-3p, TGF-β2 and FOXC1 regulating MDR in MBC and open the exploration of novel therapeutic strategies.

Purpose Breast cancer is one of the most commonly diagnosed cancers in women. Five subtypes of breast cancer differ in their genetic expression profiles and carry different prognostic values, with no treatments available for some types, such as triple-negative, due to the absence of genetic signatures that could otherwise be targeted by molecular therapies. Although endocrine treatments are largely successful for estrogen receptor (ER)-positive cancers, a significant proportion of patients with metastatic tumors fail to respond and acquire resistance to therapy. FOXA1 overexpression mediates endocrine therapy resistance in ER-positive breast cancer, although the regulation of chemotherapy response by FOXA1 has not been addressed previously. FOXA1, together with EP300 and RUNX1, regulates the expression of E-cadherin, and is expressed in luminal, but absent in triple-negative and basal-like breast cancers. We have previously determined that EP300 regulates drug resistance and tumor initiation capabilities in breast cancer cells. Methods Here we describe the generation of breast cancer cell models in which FOXA1 expression has been modulated either by expression of hairpins targeting FOXA1 mRNA or overexpression plasmids. Results Upon FOXA1 knockdown in luminal MCF-7 and T47D cells, we found an increase in doxorubicin and paclitaxel sensitivity as well as a decrease in anchorage independence. Conversely, upregulation of FOXA1 in basal-like MDA-MB-231 cells led to an increase in drug resistance and anchorage independence. Conclusion Together, these data suggest that FOXA1 plays a role in making tumors more aggressive.

Transmembrane protein with an EGF-like and two Follistatin-like domains 2 (TMEFF2) is a 374-residue long type-I transmembrane proteoglycan which is proteolytically shed from the cell surface. The protein is involved in a range of functions including metabolism, neuroprotection, apoptosis, embryonic development, onco-suppression and endocrine function. TMEFF2 is methylated in numerous cancers, and an inverse correlation with the stage, response to therapy and survival outcome has been observed. Moreover, TMEFF2 methylation increases with breast, colon and gastric cancer progression. TMEFF2 is methylated early during oncogenesis in breast and colorectal cancer, and the detection of methylated free-circulating TMEFF2 DNA has been suggested as a potential diagnostic tool. The TMEFF2 downregulation signature equals and sometimes outperforms the Gleason and pathological scores in prostate cancer. TMEFF2 is downregulated in glioma and cotricotropinomas, and it impairs the production of adrenocorticotropic hormone in glioma cells. Interestingly, through binding the amyloid β protein, its precursor and derivatives, TMEFF2 provides neuroprotection in Alzheimer's disease. Despite undergoing extensive investigation over the last two decades, the primary literature regarding TMEFF2 is incoherent and offers conflicting information, in particular, the oncogenic vs. onco-suppressive role of TMEFF2 in prostate cancer. For the first time, we have compiled, contextualised and critically analysed the vast body of TMEFF2-related literature and answered the apparent discrepancies regarding its function, tissue expression, intracellular localization and oncogenic vs. onco-suppressive role.

BackgroundChemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. MethodsSulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay ResultsTo explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1)are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10Band resistant 5-8FNPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. ConclusionCollectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells.

E-cadherin transcriptional activator EP300 is down-regulated in metaplastic breast carcinoma, a rare form of triple negative and E-cadherin-negative aggressive breast cancer with a poor clinical outcome. In order to shed light on the regulation of E-cadherin by EP300 in breast cancer we analyzed by immunohistochemistry 41 cases of invasive breast cancer with both E-cadherinhigh and E-cadherinlow expression levels, together with 20 non-malignant breast tissues. EP300 and E-cadherin showed a positive correlation in both non-malignant and cancer cases and both markers together were better predictors of lymph node metastasis than E-cadherin alone. These data support a metastasis suppressor role for EP300 in breast cancer. However, some reports suggest an oncogenic role for EP300. We generated a breast cancer cell model to study E-cadherin-independent effects of EP300 by over-expression of EP300 in HS578T cells which have E-cadherin promoter hypermethylated. In this cell system, EP300 led to up-regulation of mesenchymal (vimentin, Snail, Slug, Zeb1) and stemness (ALDH+ and CD44high/CD24low) markers, increases in migration, invasion, anchorage-independent growth and drug resistance. Genome-wide expression profiling identified aldo-keto reductases AKR1C1-3 as effectors of stemness and drug resistance, since their pharmacological inhibition with flufenamic acid restored both doxorubicin and paclitaxel sensitivity and diminished mammosphere formation. Thus, in cells with a permissive E-cadherin promoter, EP300 acts as a tumour/metastasis supressor by up-regulating E-cadherin expression, maintenance of the epithelial phenotype and avoidance of an epithelial-to-mesenchymal transition. In cells in which the E-cadherin promoter is hypermethylated, EP300 functions as an oncogene via up-regulation of aldo-keto reductases. This offers the rationale of using current aldo-keto reductase inhibitors in breast cancer treatment.

An innovative ternary copper(II) complex, [Cu(Cl‐PIP)(Tyr)Cl]n, has been synthesized and characterized using infrared spectroscopy, elemental analysis and single‐crystal X‐ray diffraction analysis. X‐ray crystallography indicates that the Cu atom is five‐coordinated in a square‐pyramidal configuration. The unit forms a one‐dimensional chain along the crystallographic c‐axis. The complex was screened for cytotoxicity against a panel of eight human cancer cell lines, namely MDA‐MB‐231, CAL‐51, K562, HeLa, SGC‐7901, A549, MCF‐7 and SMMC‐7721. The best anticancer activity was obtained with triple‐negative breast cancer CAL‐51 and MDA‐MB‐231 cell lines, with IC50 values in the range 0.035–0.10 μM, and this was better than using carboplatin. The complex inhibits proteasomal chymotrypsin‐like activity, and docking studies reveal its interaction with 20S proteasome. In addition, the complex causes accumulation of ubiquitinated proteins, induces apoptosis and inhibits cell proliferation, indicating its great potential as a novel therapy for triple‐negative breast cancer.

Five innovative ternary copper(II) complexes [Cu(OH-PIP)(Phe)Cl](1), [Cu(OH-PIP)(Gly)(H2O)]NO3·2H2O (2), [Cu(OH-PIP)(Ala)(Cl)]·H2O (3), [Cu(OH-PIP)(Met)]PF6·2H2O (4), and [Cu(OH-PIP)(Gln)(H2O)](Cl)·3H2O (5) have been synthesized and characterized by infrared spectroscopy, elemental analysis, and single crystal X-ray diffraction analysis. X-ray crystallography indicates that all Cu atoms are five-coordinated in a square-pyramidal configuration. The complexes have been screened for cytotoxicity against human breast cancer cell lines MCF-7, MDA-MB-231, and CAL-51. The best anticancer activity is obtained with triple-negative breast cancer CAL-51 and MDA-MB-231 cell lines, with IC50 values in the range of 0.082-0.69 μM. Importantly, the copper compounds were more effective than carboplatin at triggering cell death. Mechanistically, the complexes inhibit proteasomal chymotrypsin-like activity, and docking studies reveal their 20S proteasome binding sites. As a consequence, they cause the accumulation of ubiquitinated proteins, inhibit cell proliferation, and induce apoptosis. In addition, these copper complexes decrease the stemness of triple-negative breast cancer cells and have synergistic effects with CBP on TNBC cells, indicating their great potential as a novel therapy for triple-negative breast cancer.