Sustained cancer-relevant alternative RNA splicing events driven by PRMT5 in high-risk neuroblastoma.

Protein arginine methyltransferase 5 (PRMT5) is over-expressed in a wide variety of cancers and is implicated as having a key oncogenic role, achieved in part through its control of the master transcription regulator E2F1. We investigated the relevance of PRMT5 and E2F1 in neuroblastoma (NB) and found that elevated expression of PRMT5 and E2F1 occurs in poor prognosis high-risk disease and correlates with an amplified Myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) gene. Our results show that MYCN drives the expression of splicing factor genes that, together with PRMT5 and E2F1, lead to a deregulated alternative RNA splicing programme that impedes apoptosis. Pharmacological inhibition of PRMT5 or inactivation of E2F1 restores normal splicing and renders NB cells sensitive to apoptosis. Our findings suggest that a sustained cancer-relevant alternative RNA splicing programme desensitises NB cells to apoptosis, and identify PRMT5 as a potential therapeutic target for high-risk disease.

The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity.

Protein acetylation plays a key role in regulating cellular processes and is subject to aberrant control in diverse pathologies. Although histone deacetylase (HDAC) inhibitors are approved drugs for certain cancers, it is not known whether they can be deployed in other therapeutic contexts. We have explored the clinical HDAC inhibitor, zabadinostat/CXD101, and found that it is a stand-alone regulator of the adaptive immune response. Zabadinostat treatment increased expression of MHC class I and II genes in a variety of cells, including dendritic cells (DCs) and healthy tissue. Remarkably, zabadinostat enhanced the activity of DCs, and CD4 and CD8 T lymphocytes. Using an antigenic peptide presented to the immune system by MHC class I, zabadinostat caused an increase in antigen-specific CD8 T lymphocytes. Further, mice immunised with covid19 spike protein and treated with zabadinostat exhibit enhanced covid19 neutralising antibodies and an increased level of T lymphocytes. The enhanced humoral response reflected increased activity of T follicular helper (Tfh) cells and germinal centre (GC) B cells. Our results argue strongly that zabadinostat has potential to augment diverse therapeutic agents that act through the immune system.

Does memantine show chemopreventive effect against mice 4T1 breast tumor model?

BACKGROUND: Cancer cells express higher levels of N-methyl-d-aspartate (NMDA) receptor. In this study, we aimed to use memantine as a potential blocker to inhibit the action of the NMDA receptor in cancer cells in vivo in order to investigate the potential chemopreventive effect of memantine in 4T1 tumor-bearing mice. METHODS: To determine the potential chemopreventive effect of the compound, mice weights, tumor volumes, spleen IL-6, and tumor DNA methylation levels were investigated. A total of 26 Balb/c female mice were allocated into three groups. G1 (n = 6): tumor control group, G2 (n = 10): low dose (5mg/kg) memantine group, G3: high dose (10 mg/kg) memantine group (n = 10). G1 was inoculated with 4T1 cells without any memantine treatment. G2 and G3 were pretreated with 5 and 10 mg/kg memantine daily intraperitoneal (ip) injection (weekend off) for 10 days, respectively. Both G2 and G3 were subdivided into two groups as G2a (n = 4) and G3a (n = 4): tumor free groups and G2b (n = 6) and G3b (n = 6) tumor bearing groups. RESULTS: Our results revealed that G3: high dose (10 mg/kg) memantine group, significantly (p = 0.0248) reduced the tumor volumes. We found that spleen IL-6 levels were significantly higher in memantine pretreated tumor free group p = 0.0204 ) We also found that high dose memantine treated tumor free group (G3a) has significantly lower genome-wide DNA methylation levels when compared to tumor control group (G1) p = 0.0012. DISCUSSION: To the best of our knowledge, it is the first study that highlights a potential chemopreventive effect of memantine in vivo in the mouse 4T1 breast tumor model. But further investigations should be carried out to explore the chemopreventive mechanism of action for memantine in cancer.

Memantine shifts cancer cell metabolism via AMPK1/2 mediated energetic switch in A549 lung cancer cells.

Memantine is used to prevent glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease. As glutamine is one of the major source of anabolism in fast growing cancer cells, we aimed to interfere with the cancer cell metabolism in A549 lung cancer cells by using memantine. The effects of memantine on cell cycle progression and cell death in A549 cells were assessed by MTT assay and PI staining. Cells were treated with 0.25 mM memantine for 48 hours and then cell metabolism (AMPKA1, AMPKA2, HIF1A, B-catenin, PKM), apoptosis (p53, p21, Bax, Bcl-XL, NOXA, PUMA) and autophagy related (LC3B-I, LC3B-II, SQSTM1) mRNA and protein expressions were investigated by RT-qPCR and western blotting. Memantine decreased cell viability significantly in a concentration-dependent manner by inducing G0/G1 cell cycle arrest. Our results suggest that memantine activates AMPK1/2 significantly (p=0.039 and p=0.0105) that led cells through apoptosis and autophagy by decreasing cancer cell metabolism regulators like HIF1A, B-catenin and PKM as the consequence of this energetic shift. Memantine represents a useful tool to target metabolism in cancer cells. Therefore, it might be used a new repurposed drug in cancer treatment.

Immune modulation underpins the anti-cancer activity of HDAC inhibitors.

Aberrant protein acetylation is strongly linked to tumorigenesis, and modulating acetylation through targeting histone deacetylase (HDAC) with small-molecule inhibitors has been the focus of clinical trials. However, clinical success on solid tumours, such as colorectal cancer (CRC), has been limited, in part because the cancer-relevant mechanisms through which HDAC inhibitors act remain largely unknown. Here, we have explored, at the genome-wide expression level, the effects of a novel HDAC inhibitor CXD101. In human CRC cell lines, a diverse set of differentially expressed genes were up- and downregulated upon CXD101 treatment. Functional profiling of the expression data highlighted immune-relevant concepts related to antigen processing and natural killer cell-mediated cytotoxicity. Similar profiles were apparent when gene expression was investigated in murine colon26 CRC cells treated with CXD101. Significantly, these changes were also apparent in syngeneic colon26 tumours growing in vivo. The ability of CXD101 to affect immune-relevant gene expression coincided with changes in the tumour microenvironment (TME), especially in the subgroups of CD4 and CD8 tumour-infiltrating T lymphocytes. The altered TME reflected enhanced antitumour activity when CXD101 was combined with immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-CTLA4. The ability of CXD101 to reinstate immune-relevant gene expression in the TME and act together with ICIs provides a powerful rationale for exploring the combination therapy in human cancers.

Targeting Cancer Cell Metabolism with Metformin, Dichloroacetate and Memantine in Glioblastoma (GBM).

AIM: To investigate the effects of metformin, dichloroacetate (DCA), and memantine on T98G and U87-MG human glioblastoma (GBM) cells to target tumor cell metabolism in a multi-directional manner. MATERIAL AND METHODS: IC50 levels for metformin, DCA, metformin+DCA and memantine were determined by MTT assay in T98G and U87-MG cells in vitro. Casp3, Bcl-2, Bax, c-Myc and GSK-3B protein expressions were investigated post treatments. Fifteen GBM+ tumor tissues were assessed for Casp-3, Bcl-2, Bad, Bax for apoptotic protein expression patterns. RESULTS: Cancer cell metabolism targeting drugs metformin, DCA, metformin+DCA and memantine induced cytotoxicity in a dose-dependent manner in T98G and U87-MG cells. IC50 for memantine is found as 0.5 mM (p < 0.01) which is nearly 10 times lower concentration than that of metformin. Fifteen GBM+ tumor tissues had differential apoptotic protein expressions. CONCLUSION: Memantine exerted anti-cancer mechanism of action in T98G and U87-MG cells, however, such a mechanism requires deeper investigation for GBM treatment.

Antitumorigenic Effect of Memantine via Interfering Glutamate Metabolism in Mouse 4T1 Breast Tumor Model.

BACKGROUND: Repurposing drug is an efficient strategy as the drug discovery process is timeconsuming, laborious and costly. Memantine is already used in Alzheimer's disease to prevent neurons from excess glutamate toxicity. As cancer cells benefit from higher amounts of cellular energetics like glucose and glutamine, we used memantine to interfere with the glutamate metabolism in order to restrict cancer cells' glutamine as a source for their growth. OBJECTIVE: To investigate the potential antitumor effect of memantine by reducing glutamate levels in 4T1 mouse breast cancer model. METHODS: 24 Balb/c female mice were subcutaneously inoculated with 4T1 cells. When tumors were palpable, memantine treatment was initiated as 5 and 10 mg/kg daily intraperitoneal injection. Tumor growth was recorded every 2-3 days. Tumor volumes, serum glutamate levels, spleen IL-6 levels, genome-wide DNA methylation levels and GSK3B. pGSK3B protein expressions were measured to enlighten the anticancer mechanism of action for memantine. RESULTS: We found that both two doses (5 and 10mg/kg) decreased tumor growth rates and serum glutamate levels significantly (p<0.05). 10mg/kg treatment increased spleen IL-6 levels (p<0.05) and decreased genomewide DNA methylation levels. Memantine treatment decreased GSK3B protein expression levels in tumor tissue samples. CONCLUSION: To the best of our knowledge, this is the first study that investigates the antitumor activity of memantine in a breast cancer tumor model. Our results suggest a potent anticancer mechanism of the action for memantine. Memantine decreased genome wide methylation and serum glutamate levels that are associated with a poor prognosis. Therefore, Memantine might be used for targeting glutamine metabolism in cancer treatment.

The Importance of Cancer Stem Cells in Gastrointestinal Cancers.

Cancer is a major cause of death worldwide. Gastrointestinal (GI) cancers are still an important cause of tumor-related deaths. Conventional therapeutic approaches like surgery, chemotherapy, and radiation therapy are indispensable but insufficient. Despite the advances in immunotherapy and other conventional treatment strategies, some might argue that we are still far from curing cancer. Cancer stem cells (CSCs) might be to blame. Because sequencing technologies are moving fast in this precision-medicine era, molecular testing of tumors, circulating tumor cells, and CSCs have important implications for treatment decisions. Biomarkers offer prognostic values and targeted therapy availabilities as molecular predictive factors. CSCs are considered biomarkers of tumorigenesis development, metastasis, and recurrence. In this review, we summarize the important issues over CSCs and the development of new drugs that target GI CSCs.

The outcomes of an impaired powerhouse in KRAS mutant lung adenocarcinoma cells by Elesclomol.

OBJECTIVES: Lung cancer stands out as the most common cancer type worldwide. The most common genetic alteration detected in adenocarcinoma patients is KRAS. KRAS mutated patients still cannot get benefit from precision medicine approaches and lack a targeted therapy. Elesclomol is an investigational agent for melanoma and other malignancies. In this study, we evaluated its effect on cellular apoptosis, survival, and metastasis mechanisms on KRAS mutant A549 and Calu-1 cell lines. METHODS: The cytotoxic effects of Elesclomol on A549 and Calu-1 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability test. Cells were treated with IC50 concentration and then apoptosis-related (Casp-3, Casp-9, Bcl-2, and Bcl-xL), survival-related (Akt, p-Akt, Erk, and p-Erk), and metastasis-related (E-cadherin, Vimentin, MMP-2, and MMP-9) protein expressions were determined by Western blot analysis. Elesclomol's effect on cell migration was evaluated by wound healing. Total oxidant, malondialdehyde (MDA), and glutathione (GSH) levels after Elesclomol treatment were assessed. RESULTS: Elesclomol not only induced apoptotic proteins but also inhibited metastatic protein expressions and migration in both cells. Also, p-Erk activity was diminished by Elesclomol treatment as a reflection of decreased proliferation. However, p-Akt was enhanced as a cellular survival mechanism. Although Elesclomol's effects on oxidative stress parameters were puzzling, it induced total oxidant status (TOS), and MDA in Calu-1 cells. CONCLUSION: Elesclomol might provide an alternative treatment approach for patients with KRAS mutant lung adenocarcinoma and other solid tumor malignancies that harbor KRAS mutations. This would enable the development of biomarker-driven targeted therapy for KRAS mutant adenocarcinoma patients.

FOXA1 knock-out via CRISPR/Cas9 altered Casp-9, Bax, CCND1, CDK4, and fibronectin expressions in LNCaP cells.

Prostate cancer is one of the most common types of cancer in men and the leading cause of death in developed countries. With the aid of molecular and genetic profiling of cancers, cancer molecular subtypes are paving the way for tailored cancer therapy. FOXA1 has been identified as one of the seven molecular subtypes of prostate cancer. FOXA1 is involved in a variety of metabolic process such as glucose homeostasis and deregulation of its expression is crucial in prostate cancer progression. In this study, we investigated the effects of FOXA1 gene knock-out on the expression levels of various cancer cell metabolism and cell cycle-related protein expressions. FOXA1 gene was knocked-out by using CRISPR/Cas9 technique. While FOXA1 gene knock-out significantly altered Casp-9, Bax, CCND1, CDK4, and fibronectin protein expressions (P < 0.05, fold change: ∼40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, ß-catenin, c-Myc, and GSK-3B. Knocking-out FOXA1 gene in androgen-dependent LNCaP prostate cancer cells inhibited CCND1 protein expression. Our pre-clinical results demonstrate the importance of FOXA1 as a drug target in the treatment of prostate cancer. Impact statement Knock-out studies offer a unique way of studying the function of genes especially for developmentally lethal genes. FOXA1 has prominent roles both in breast and prostate cancer pathogenesis due to its role in ER receptor signaling pathway. FOXA1 has also been identified as one of the seven molecular subtypes of primary prostate cancer. In the present study, we used an efficient gene knock-out method, CRISPR/Cas9, in order to investigate FOXA1 function on LNCaP prostate cancer cells in vitro. FOXA1 knock-out altered cell-cycle regulator CCND1 protein expression levels. Therefore, our results suggest that FOXA1 might be a plausible drug target for prostate cancer treatment.

Do the expressions of epithelial-mesenchymal transition proteins, periostin, integrin-α4 and fibronectin correlate with clinico-pathological features and prognosis of metastatic castration-resistant prostate cancer?

Development of metastatic castration-resistant prostate cancer is a result of the lack of an apoptotic response by the tumor cells and loss of the ability to stick to adjacent cells through epithelial-mesenchymal transition. Although there are several strongly recommended biomarkers for determining prognosis of metastatic castration-resistant prostate cancer, only few of them may help decide the selection of the optimal treatment option. The mode of treatment sequencing in metastatic castration-resistant prostate cancer will be based on the individual characteristics of the patient. In this study, we aimed to explain the correlation between the expression characteristics of periostin, integrin-α4, and fibronectin in metastatic castration-resistant prostate cancer patients and their clinico-pathological data comprising Gleason score, PSA levels, and metastatic sites in the process of epithelial-mesenchymal transition. We evaluated by using Western blotting, periostin, integrin-α4, and fibronectin expressions in peripheral blood samples of metastatic castration-resistant prostate cancer patients ( n = 40), benign prostatic hyperplasia patients ( n = 20), and the healthy control group ( n = 20). Associations between changes in the protein expressions and clinico-pathological parameters were also analyzed in the metastatic castration-resistant prostate cancer group. When comparing BPH and healthy groups with the metastatic castration-resistant prostate cancer group, a reduced expression of integrin-α4 was found in metastatic patients, albeit being statistically insignificant ( P > 0.05). Protein expressions of periostin and fibronectin in the metastatic castration-resistant prostate cancer group were higher than those in the BPH and heathy groups ( P < 0.001). Increased periostin expression in metastatic patients was significantly associated with bone metastasis ( P < 0.05). Elevated periostin and fibronectin levels in metastatic castration-resistant prostate cancer patients may be appropriate targets of therapeutic intervention in the future. Impact statement Prostate cancer is the third most common cancer in the world and the most common cancer among men. Development of metastatic castration-resistant prostate cancer (mCRPC) is a result of the lack of an apoptotic response by the tumor cells and loss of the ability to stick to adjacent cells through epithelial-mesenchymal transition (EMT). The present study analyzes for the first time the expressions of EMT marker proteins - periostin, integrin α4, fibronectin - in mCRPC and in benign prostatic hyperplasia (BPH) with the aim to determine the clinical relevance of changes in these three proteins vis-a-vis the PCa aggressive phenotype. In doing so, it sheds light on the molecular mechanism underlying the disease. We concluded that elevated periostin and fibronectin levels in mCRPC patients may be appropriate targets of therapeutic intervention in the future; hence, adopting methods that target these proteins may help treat prostate cancer effectively.