Aggressive Cancer Cells Research Articles

The Influence of Extracts from Common Houseleek (Sempervivum tectorum) on the Metabolic Activity of Human Melanoma Cells WM-266-4

Human melanoma cells are known as one of the most aggressive cancer cells, and consequently, melanoma is one of the most incurable cancer diseases. There is intense activity in research and development of potential medicines for malignant diseases, including alternative forms of remedies. Therefore, the purpose of our work was testing extracts from the common houseleek (Sempervivum tectorum) grown in Slovenia to establish its impact on human melanoma cells. Namely, we wanted to verify if the extracts inhibit growth of malignant cells and their metabolic activity. Soxhlet, cold solvent, ultrasound, and supercritical extraction methods were applied to obtain S. tectorum extracts. Polyphenols and proanthocyanins content in acquired extracts was determined as well as their antioxidative potential. For a relevant comparison, Chinese (CHI) dried and Slovenian (SLO) lyophilized S. tectorum was used. Results showed that the highest contents of polyphenols and proanthocyanins were yielded from lyophilized material, which also had the highest antioxidative potential. The focus of our work was on analysis of possible inhibition effects of the extracts on human melanoma cells since no past studies were found regarding the possible effects of S. tectorum on metabolic activity of WM-266-4. We established that in a 24-h incubation period, the extracts inhibited metabolic activity of the cells at their concentrations of 20, 10, 4, 2, 1, 0.2, and 0.02 mg/mL. Extract obtained from SLO S. tectorum (ultrasound extraction with acetone as a solvent), which showed promising results of inhibitory effect on the mentioned cells, was further described since the local plant was the focus of our study. CHI S. tectorum extract (Soxhlet extraction with ehtanol:water mixture = 1:1 as a solvent) showed the highest inhibitory effect on human melanoma cells WM-266-4, although both obtained extracts are suitable for their growth inhibition of this specific cell line. Our results suggest inhibitory ability of S. tectorum extracts on the metabolic activity of WM-266-4 metastatic cell line, suggesting their potential use as an anticancer agent.

RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers.

Metastases are the leading cause of death in cancer patients. RhoC, a member of the Rho GTPase family, has been shown to facilitate metastasis of aggressive breast cancer cells by influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. A common phenotypic characteristic of metastatic cells influenced by these mechanisms is dysregulation of cell-cell junctions. Thus, we set out to study how RhoA- and RhoC-GTPase influence the cell-cell junctions in aggressive breast cancers. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM 149 and MDA 231 breast cancer cells results in increased normalization of junctional integrity denoted by junction protein expression/colocalization. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity and increased cell-cell adhesion compared to wild-type cells. Whole exome RNA sequencing and targeted gene expression profiling demonstrate decreased expression of Type I interferon-stimulated genes in RhoC knockout cells compared to wild-type, and subsequent treatment with interferon-alpha resulted in significant increases in adhesion and decreases in invasiveness of wild-type cells and a dampened response to interferon-alpha stimulation with respect to adhesion and invasiveness in RhoC knockout cells. We delineate a key role of RhoC-GTPase in modulation of junctions and response to interferon, which supports inhibition of RhoC as a potential anti-invasion therapeutic strategy.

Betulin Sulfonamides as Carbonic Anhydrase Inhibitors and Anticancer Agents in Breast Cancer Cells.

Hypoxia-regulated protein carbonic anhydrase IX (CA IX) is up-regulated in different tumor entities and correlated with poor prognosis in breast cancer patients. Due to the radio- and chemotherapy resistance of solid hypoxic tumors, derivatives of betulinic acid (BA), a natural compound with anticancer properties, seem to be promising to benefit these cancer patients. We synthesized new betulin sulfonamides and determined their cytotoxicity in different breast cancer cell lines. Additionally, we investigated their effects on clonogenic survival, cell death, extracellular pH, HIF-1α, CA IX and CA XII protein levels and radiosensitivity. Our study revealed that cytotoxicity increased after treatment with the betulin sulfonamides compared to BA or their precursors, especially in triple-negative breast cancer (TNBC) cells. CA IX activity as well as CA IX and CA XII protein levels were reduced by the betulin sulfonamides. We observed elevated inhibitory efficiency against protumorigenic processes such as proliferation and clonogenic survival and the promotion of cell death and radiosensitivity compared to the precursor derivatives. In particular, TNBC cells showed benefit from the addition of sulfonamides onto BA and revealed that betulin sulfonamides are promising compounds to treat more aggressive breast cancers, or are at the same level against less aggressive breast cancer cells.

M6A Methyltransferase 3 Promotes the Proliferation and Migration of Gastric Cancer Cells through the m6A Modification of YAP1.

Gastric cancer is the most common gastrointestinal tumor with an increasing incidence. Furthermore, advanced gastric cancer is more common, but the mechanism underlying the proliferation and metastasis of gastric cancer has not been thoroughly explored. N6-methyladenosine (m6A) methyltransferase 3 (METTL3) may be involved in the proliferation and metastasis of gastric cancer. Therefore, Yes-associated protein 1 (YAP1) in the Hippo pathway was selected as the target, and the relationship between METTL3 and the proliferation and metastasis of gastric cancer was proved through a series of experiments. This research showed that the expression of m6A and METTL3 was upregulated in human gastric cancer tissues and gastric cancer cell lines. After lentiviral transfection, METTL3 silencing in AGS (human gastric adenocarcinoma cell line AGS) and MKN-45 (human gastric cancer cell line MKN-45) gastric cancer cell lines directly inhibited the proliferation, aggressiveness, and migration of gastric cancer cells. Mechanically, the inhibition of the YAP1-TEAD signaling pathway by peptide 17 reduces m6A methylation and the total mRNA level of YAP1. It also eliminates the promoting effect of METTL3 on the proliferation and migration of gastric cancer cells. In turn, the overexpression of YAP1 eliminates the inhibitory effect of METTL3 silencing on the proliferation, migration, and invasion of gastric cancer cells. This article proved that m6A methyltransferase METTL3 promoted the proliferation and migration of gastric cancer cells through the m6A modification of YAP1.

Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis.

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule-cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell-mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid "shields" against self-destruction.

Hyaluronic acid-functionalized halloysite nanotubes for targeted drug delivery to CD44-overexpressing cancer cells

Chemotherapeutic drug has inevitable drawbacks of nonspecific tumor targeting and poor therapeutic efficiency, limiting practical clinical applications. To overcome these challenges, a novel halloysite nanotube-based drug delivery system was designed for efficient CD44-mediated targeting in this study. CD44 molecule, a major receptor for hyaluronic acid (HA), overexpressed in aggressive cancer cells. The successful synthesis of hyaluronic acid modified halloysite (HNTs-NH-HA) complexes were proved by 13C solid state NMR spectroscopy, FT-IR, XPS and TGA. Thereafter, a model anticancer drug doxorubicin (DOX) was loaded into HNTs-NH-HA to from the final drug delivery system (HNTs-NH-HA/DOX). In vitro cytotoxicity assay tests revealed that HNTs-NH-HA/DOX improved therapeutic efficacy of DOX by specific targeting. Furthermore, confocal laser scanning microscopy (CLSM) results showed better uptake of HNTs-NH-HA/DOX nanoparticles in CD44 receptor-positive cells than in CD44 receptor-negative cells. It was expected that HNTs-NH-HA drug delivery systems hold great potential for targeting CD44 over-expressing tumors.

Blockade of CHRNB2 signaling with a therapeutic monoclonal antibody attenuates the aggressiveness of gastric cancer cells.

Here, we evaluated the therapeutic potential of antibodies (Abs) targeting cholinergic receptor nicotinic beta 2 subunit (CHRNB2) in gastric cancer. To investigate the effects of these Abs on malignant phenotypes in vitro and in mouse xenograft models, we generated gene knockouts through genome editing, performed RNA interference-mediated knockdown of gene expression, and ectopically expressed CHRNB2 in gastric cancer cells. The effects of anti-CHRNB2 Abs on the proliferation of cancer cells were evaluated both in vitro and in vivo. We determined the effects of Chrnb2 deficiency on mice and the clinical significance of CHRNB2 expression in gastric cancer clinical specimens. Knockdown of CHRNB2 attenuated gastric cancer cell proliferation, whereas forced overexpression of CHRNB2 increased cell proliferation. Knockout of CHRNB2 significantly influenced cell survival and functions associated with metastasis. The effects of polyclonal Abs targeting the C- and N-termini of CHRNB2 guided the development of anti-CHRNB2 monoclonal Abs that inhibited the growth of gastric cancer cells in vitro and in vivo. Pathway analysis revealed that CHRNB2 interfered with signaling through the PI3K-AKT and JAK-STAT pathways. Chrnb2-deficient mice exhibited normal reproduction, organ functions, and motor functions. CHRNB2 regulates multiple oncological phenotypes associated with metastasis, and blockade of CHRNB2 expression using specific Abs shows promise for controlling metastasis in gastric cancer.

Quantitative Capillary Electrophoresis for Analysis of Extracellular Vesicles (EVqCE)

Extracellular Vesicles (EVs) gained significant interest within the last decade as a new source of biomarkers for the early detection of diseases and a promising tool for therapeutic applications. In this work, we present Extracellular Vesicles Quantitative Capillary Electrophoresis (EVqCE) to measure an average mass of RNA in EVs, determine EV concentrations and the degree of EV degradation after sample handling. We used EVqCE to analyze EVs isolated from conditioned media of three cancer cell lines. EVqCE employs capillary zone electrophoresis with laser-induced fluorescent detection to separate intact EVs from free nucleic acids. After lysis of EVs with a detergent, the encapsulated nucleic acids are released. Therefore, the initial concentration of intact EVs is calculated based on a nucleic acid peak gain. EVqCE works in a dynamic range of EV concentrations from 108 to 1010 particles/mL. The quantification process can be completed in less than one hour and requires minimum optimization. Furthermore, the average mass of RNA was found to be in the range of 200–400 ag per particle, noting that more aggressive cancer cells have less RNA in EVs (200 ag per particle) than non-aggressive cancer cells (350 ag per particle). EVqCE works well for the degradation analysis of EVs. Sonication for 10 min at 40 kHz caused 85% degradation of EVs, 10 freeze-thaw cycles (from −80 °C to 22 °C) produced 40%, 14-day storage at 4 °C made 32%, and vortexing for 5 min caused 5% degradation. Presently, EVqCE cannot separate and distinguish individual EV populations (exosomes, microvesicles, apoptotic bodies) from each other. Still, it is tolerant to the presence of non-EV particles, protein-lipid complexes, and protein aggregates.

Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.

Influence of nNav1.5 on MHC class I expression in breast cancer

Cancer metastasis occurs due to the ability of the tumour to evade immune recognition and response by altering the major histocompatibility complex class I (MHC class I). A comprehensive understanding of the MHC class I alteration in breast cancer metastasis is vital for the rational design and improvement of immunotherapies for advanced cancer. In this study, we associate it with the elevation of 'neonatal' Nav1.5 (nNav1.5) mRNA expression in the aggressive type of human breast cancer cells. Using real-time PCR, lower expression of TAP1 (by - 4.83-fold), which is a part of the MHC class I antigen processing machinery (APM) pathway component, in contrast to the greater expression of nNav1.5 (by 3.76-fold), along with other metastatic markers, MMP1 (by 57.48-fold) and fibronectin (by 2.88-fold) in aggressive human breast cancer cell line, MDA-MB231 were obtained. Subsequent knockdown of nNav1.5 (by 52.6%) in MDA-MB-231 via siRNA-SCN5A resulted in downregulation of another metastatic marker, fibronectin (by 52.9%) but importantly, rescued MHC class I expression (by 347% or 3.47-fold). Furthermore, the siRNA-SCN5A transfected cells failed to form a unified spheroid and incapable of efficiently invading the surrounding invasion matrix. In summary, the influence of nNav1.5 in regulating MHC class I mRNA expression to allow for breast cancer invasion is demonstrated, supporting the potential of nNav1.5 as an immunotherapy target to overcome tumour immune evasion thus preventing metastasis.

Effects of photobiomodulation by low power lasers on the in vitro proliferation and aggressiveness of breast cancer cells

Through the metastatic process, cancer cells spread through the body by migrating and invading via the extracellular matrix. Photobiomodulation therapy (PBMT) on oncologic patients is controversial as it could stimulate cell proliferation and aggravate the metastatic process. Thus, this study aimed to investigate PBMT induced by low power red and infrared lasers on cell proliferation, migration and invasion and on mRNA levels from Rat sarcoma virus (RAS) oncogenes in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were irradiated with low power red and infrared lasers at 25 and 50 J cm−2, cell proliferation was evaluated using water-soluble tetrazolium salt assay, cell migration was evaluated using wound healing assay, cell invasion was performed using Matrigel transwell assay and real-time quantitative polymerase chain reaction was performed to evaluate mRNA of RAS oncogenes. Exposure to low power infrared laser could increase MDA-MB-231 cell proliferation at 25 J cm−2. Neuroblastoma RAS oncogene (NRAS) mRNA relative levels reduced when irradiating with low power infrared at 25 J cm−2 and for low power red laser at 50 J cm−2. Kirsten rat sarcoma viral oncogene (KRAS) mRNA relative levels were not modified. Cell migration and invasion after exposure to low power red and infrared lasers at both fluences evaluated did not change. These results show that PBMT could modify MDA-MB-231 cell proliferation and NRAS mRNA relative levels, but no effects were found on KRAS mRNA relative levels, cell migration and invasion in both red and infrared lasers. Therefore, the screening for therapies should be tested by searching for safe conditions and herein the PBMT using red lasers altered the proliferation ratio and changed the aggressiveness phenotype of breast cancer cells.

MIER3 induces epithelial-mesenchymal transition and promotes breast cancer cell aggressiveness via forming a co-repressor complex with HDAC1/HDAC2/Snail

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer death in women. MIER3 (Mesoderm induction early response 1, family member3) is considered as a potential oncogene for breast cancer. However, the role of MIER3 in breast cancer remain largely unknown. The expression of MIER3 was detected and the relationship between its expression and clinicopathological characteristics was also analyzed. The effect of MIER3 on proliferation and migration of breast cancer cells was detected in vitro and in vivo. Western blot, IF, and Co-IP were employed to detect the relationship between MIER3, HDAC1, HDAC2, and Snail. ChIP assay was performed to determine the binding of MIER3/HDAC1/HDAC2/Snail complex to the promoter of E-cadherin. In this study, we found that MIER3 was upregulated in breast cancer tissue and closely associated with poor prognosis of patients. MIER3 could promote the proliferation, migration, and epithelial-mesenchymal transition (EMT) of breast cancer cells. Further studies showed that MIER3 interacted with HDAC1/HDAC2 and Snail to form a repressive complex which could bind to E-cadherin promoter and was related to its deacetylation. Our study concluded that MIER3 was involved in forming a co-repressor complex with HDAC1/HDAC2/Snail to promote EMT by silencing E-cadherin.

Extracellular Vesicles as Mediators of Cancer Disease and as Nanosystems in Theranostic Applications.

Simple SummaryCancer is the second leading cause of death in humans, and in 2020, 9.8 million cancer-related deaths were reported worldwide. In the last 20 years, it has become apparent that small vesicles released by cancer cells, referred to as extracellular vesicles (EVs), are key players in cell–cell communication in the tumor environment, and as a consequence, research in this area has increased dramatically. This review summarizes the recent advances in our understanding of how EVs serve as mediators of communication between cancer cells and with their surroundings in order to promote the acquisition of specific characteristics that permit their aberrant behavior. In addition, we dwell on how EVs aid in the development of drug resistance, which is a frequent cause of treatment failure in chemotherapy. Finally, we discuss an exciting new area of research that envisions harnessing the unique characteristics of EVs for therapeutic and diagnostic purposes (theranostics). Taken together, the available literature suggests that advances in our understanding of EV biology in the next decades will likely be critical to achieving more effective treatments in cancer patients.Cancer remains a leading cause of death worldwide despite decades of intense efforts to understand the molecular underpinnings of the disease. To date, much of the focus in research has been on the cancer cells themselves and how they acquire specific traits during disease development and progression. However, these cells are known to secrete large numbers of extracellular vesicles (EVs), which are now becoming recognized as key players in cancer. EVs contain a large number of different molecules, including but not limited to proteins, mRNAs, and miRNAs, and they are actively secreted by many different cell types. In the last two decades, a considerable body of evidence has become available indicating that EVs play a very active role in cell communication. Cancer cells are heterogeneous, and recent evidence reveals that cancer cell-derived EV cargos can change the behavior of target cells. For instance, more aggressive cancer cells can transfer their “traits” to less aggressive cancer cells and convert them into more malignant tumor cells or, alternatively, eliminate those cells in a process referred to as “cell competition”. This review discusses how EVs participate in the multistep acquisition of specific traits developed by tumor cells, which are referred to as “the hallmarks of cancer” defined by Hanahan and Weinberg. Moreover, as will be discussed, EVs play an important role in drug resistance, and these more recent advances may explain, at least in part, why pharmacological therapies are often ineffective. Finally, we discuss literature proposing the use of EVs for therapeutic and prognostic purposes in cancer.

Abstract 2838: Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state

Abstract Epigenetic dysregulations have a recognized role in cancer pathogenesis. Histone methyltransferase MLL3 (mixed-lineage leukemia protein 3) (gene symbol - KMT2C) is one of the most frequently altered genes in human breast cancer. In addition to the loss-of-function mutations, the expression levels of MLL3 are downregulated significantly in the tumor, compared to normal breast samples. Consistent with these cancer genome findings, functional studies in mouse models and human cancer cells showed loss of MLL3 promotes tumor initiation and growth. However, the role of MLL3 mutation in metastasis has not been determined. Here we showed that the MLL3 levels were further reduced in distant metastases compared to primary tumors, suggesting a potential role of MLL3 loss in metastasis. We demonstrate that MLL3 loss markedly promoted distant metastasis through specifically enhancing the step of colonization during the metastatic cascade. MLL3 loss facilitated the acquisition of an epithelial/mesenchymal (E/M) transition state by cancer cells disseminated to the lung. These cells have a hybrid phenotype of epithelial and mesenchymal features. We further showed that the E/M transitional cells induced by MLL3 loss have greatly increased tumor-initiating ability and multi-organ metastatic ability. We further demonstrated that MLL3 lose facilitated luminal breast tumor cells to acquire the E/M hybrid features and promoted endocrine therapy resistance, suggesting that loss of MLL3 also promotes the epithelial-mesenchymal plasticity and therapeutic resistance. To identify vulnerability of MLL3-mutant cancer cells, we performed small molecule screening and discovered that MLL3-mutant cancer cells are markedly more sensitive to BET inhibitors than MLL3-WT cells. The in vivo study confirmed that the BET inhibitor repressed the MLL3-mutant tumor growth much more efficiently than MLL3-WT tumors. Furthermore, the BET inhibitor is effective in suppressing the growth of established MLL3-mutant lung metastasis. Taken together, our findings indicate that MLL3 mutations promotes metastasis and therapeutic resistance by enhancing epithelial-mesenchymal plasticity. However, these aggressive cancer cells are uniquely vulnerable to BET inhibition, suggesting a potential therapeutic strategy for MLL3-mutant cancers. Citation Format: Jihong Cui, Kai Ge, Wenjun Guo. Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2838.

Abstract 2060: Therapeutic approaches for CCNE1-amplified HR-proficient ovarian cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have shown great success in ovarian cancer patients that are homologous recombination deficient (HRD). However, approximately 50% of high grade serous ovarian cancer (HGSOC) are HR-proficient. HR-proficient Cancer cells evade lethal DNA damage by triggering HR pathways at the cost of genome stability and evolving of more aggressive cancer cells. Cyclin E1 (CCNE1) amplification is a major subset of known HR-proficient HGSOC. This subset of cancer responds poorly to essentially all available ovarian cancer therapies including PARP inhibitors. Our study showed that the mTOR, HR, and checkpoint pathways were enriched in CCNE1-amplifed ovarian cancer patients. Interactome mapping demonstrated that many of the interactive proteins in the mTOR pathway were also components of HR and DNA checkpoint pathways. mTOR inhibition resulted in downregulation of HR and DNA checkpoint proteins. Downregulation of ATR partly mirrored mTOR inhibition. mTOR inhibitors and ATR inhibitors synergized with PARP inhibitors in vitro and in vivo in CCNE1-amplified HGSOC tumors. This was associated with decrease in cancer stem-like cancer cells and loss of polyploid tumor cells, which are vital for evolving of more aggressive, therapy-resistant cancer cells. These studies will have potential impact on further preclinical validation and clinical applications to cure CCNE1-amplifed HRP ovarian cancer. Citation Format: Shoumei Bai, Ronald J. Buckanovich. Therapeutic approaches for CCNE1-amplified HR-proficient ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2060.

Abstract 975: Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer

Abstract Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype and c-Myc amplification has been shown to be frequent in esophageal cancer. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. We therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a CDK inhibitor flavopiridol. We found frequent overexpression of c-Myc in esophageal cancer cell lines and tissues. c-Myc overexpression correlated with accelerated esophageal cancer subcutaneous xenograft tumor growth without association with improved overall survival in esophageal cancer patients. c-Myc expression was enhanced by Wnt signaling activator (6-bromoindirubin-3′ -oxime (BIO) or pcDNA3-cMyc and c-Myc expression was depleted by c-Myc siRNAs. Interestingly, esophageal cancer cells with elevated c-Myc expression were preferentially more sensitive to induction of apoptosis by CDK inhibition flavopiridol compared to esophageal cancer cells with lower c-Myc expression. In addition, we observed that flavopiridol alone or in combinations with chemotherapeutic agent nanoparticle albumin-bound paclitaxel (NPT) or in combinations with targeted agent BMS-754807 significantly inhibited esophageal cancer cell proliferation and subcutaneous xenograft tumor growth with significantly enhancing overall mice survival. These results indicate that aggressive esophageal cancer cells with elevated c-Myc expression are sensitive to CDK inhibitor flavopiridol and flavopiridol alone or in combination with cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing esophageal cancer. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Jun Li, Margaret A. Schwarz, Urs von Holzen. Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 975.

Epigenetic Upregulation of MAGE-A Isoforms Promotes Breast Cancer Cell Aggressiveness.

Simple SummaryBreast cancer is a heterogeneous disease that has complex causes and mechanisms of development. Currently, patient treatment options depend on the breast cancer molecular subtype, which is classified based on the presence or absence of hormone receptors and HER2. However, this classification system has limitations in terms of predicting responsiveness to anticancer drugs and patient outcomes. In this study, we present a new approach to classifying molecular breast cancer subtypes: it is based on changes in histone modifications in the promoter region of the MAGEA12 locus, which we found related closely to MAGEA12 expression and MAGEA12-associated malignancy of breast cancer cells.After decades-long efforts to diagnose and treat breast cancer, the management strategy that has proved most successful to date is molecular-subtype-specific inhibition of the hormone receptors and HER2 that are expressed by individual cancers. Melanoma-associated antigen (MAGE) proteins comprise >40 highly conserved members that contain the MAGE homology domain. They are often overexpressed in multiple cancers and contribute to cancer progression and metastasis. However, it remains unclear whether the biological activity arising from MAGE gene expression is associated with breast cancer subtypes. In this study, we analyzed the RNA-sequencing (RNA-seq) data of 70 breast cancer cell lines and found that MAGEA12 and MAGEA3 were highly expressed in a subset of these lines. Significantly, MAGEA12 and MAGEA3 expression levels were independent of hormone receptor expression levels but were closely associated with markers of active histone modifications. This indicates that overexpression of these genes is attributable to epigenetic deregulation. RNA-seq of MAGEA12-depleted cells was then used to identify 382 candidate targets of MAGEA12 that were downregulated by MAGEA12 depletion. Furthermore, our gain-of-function experiments showed that MAGEA12 overexpression promoted aggressive behaviors of malignant breast cancer cells, including enhancing their cell migration and invasion. These changes were associated with increased epigenetic deregulation of the MAGEA12 signature genes. Thus, MAGEA12 may play an important role in breast cancer malignancy. Taken together, our findings suggest that MAGEA12 could be a promising therapeutic target in breast cancer, and its overexpression and epigenetic changes could serve as subtype classification biomarkers.

Targeting Pyruvate Kinase M2 Phosphorylation Reverses Aggressive Cancer Phenotypes.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low survival rate and a lack of biomarkers and targeted treatments. Here, we target pyruvate kinase M2 (PKM2), a key metabolic component of oncogenesis. In patients with TNBC, PKM2pS37 was identified as a prominent phosphoprotein corresponding to the aggressive breast cancer phenotype that showed a characteristic nuclear staining pattern and prognostic value. Phosphorylation of PKM2 at S37 was connected with a cyclin-dependent kinase (CDK) pathway in TNBC cells. In parallel, pyruvate kinase activator TEPP-46 bound PKM2pS37 and reduced its nuclear localization. In a TNBC mouse xenograft model, treatment with either TEPP-46 or the potent CDK inhibitor dinaciclib reduced tumor growth and diminished PKM2pS37. Combinations of dinaciclib with TEPP-46 reduced cell invasion, impaired redox balance, and triggered cancer cell death. Collectively, these data support an approach to identify PKM2pS37-positive TNBC and target the PKM2 regulatory axis as a potential treatment. SIGNIFICANCE: PKM2 phosphorylation marks aggressive breast cancer cell phenotypes and targeting PKM2pS37 could be an effective therapeutic approach for treating triple-negative breast cancer.

Vimentin Promotes the Aggressiveness of Triple Negative Breast Cancer Cells Surviving Chemotherapeutic Treatment

Tremendous data have been accumulated in the effort to understand chemoresistance of triple negative breast cancer (TNBC). However, modifications in cancer cells surviving combined and sequential treatment still remain poorly described. In order to mimic clinical neoadjuvant treatment, we first treated MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide for 2 days, and then with paclitaxel for another 2 days. After 4 days of recovery, persistent cells surviving the treatment were characterized at both cellular and molecular level. Persistent cells exhibited increased growth and were more invasive in vitro and in zebrafish model. Persistent cells were enriched for vimentinhigh sub-population, vimentin knockdown using siRNA approach decreased the invasive and sphere forming capacities as well as Akt phosphorylation in persistent cells, indicating that vimentin is involved in chemotherapeutic treatment-induced enhancement of TNBC aggressiveness. Interestingly, ectopic vimentin overexpression in native cells increased cell invasion and sphere formation as well as Akt phosphorylation. Furthermore, vimentin overexpression alone rendered the native cells resistant to the drugs, while vimentin knockdown rendered them more sensitive to the drugs. Together, our data suggest that vimentin could be considered as a new targetable player in the ever-elusive status of drug resistance and recurrence of TNBC.

The Multifaceted Functions of Autophagy in Breast Cancer Development and Treatment.

Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by the formation of double-membrane vesicles called autophagosomes. During this process, autophagosomes engulf and deliver their intracellular content to lysosomes, where they are degraded by hydrolytic enzymes. Thereby, autophagy provides energy and building blocks to maintain cellular homeostasis and represents a dynamic recycling mechanism. Importantly, the clearance of damaged organelles and aggregated molecules by autophagy in normal cells contributes to cancer prevention. Therefore, the dysfunction of autophagy has a major impact on the cell fate and can contribute to tumorigenesis. Breast cancer is the most common cancer in women and has the highest mortality rate among all cancers in women worldwide. Breast cancer patients often have a good short-term prognosis, but long-term survivors often experience aggressive recurrence. This phenomenon might be explained by the high heterogeneity of breast cancer tumors rendering mammary tumors difficult to target. This review focuses on the mechanisms of autophagy during breast carcinogenesis and sheds light on the role of autophagy in the traits of aggressive breast cancer cells such as migration, invasion, and therapeutic resistance.