Tetrandrine for Targeting Therapy Resistance in Cancer.

During the last five decades, there has been tremendous development in our understanding of cancer biology and the development of new and novel therapeutics to target cancer. However, despite these advances, cancer remains the second leading cause of death across the globe. Most cancer deaths are attributed to the development of resistance to current therapies. There is an urgent and unmet need to address cancer therapy resistance. Tetrandrine, a bis-benzyl iso-quinoline, has shown a promising role as an anti-cancer agent. Recent work from our laboratory and others suggests that tetrandrine and its derivatives could be an excellent adjuvant to the current arsenal of anti-cancer drugs. Herein, we provide an overview of resistance mechanisms to current therapeutics and review the existing literature on the anti-cancer effects of tetrandrine and its potential use for overcoming therapy resistance in cancer.

Regulation of SPDEF expression by DNA methylation in advanced prostate cancer.

Introduction: Prostate cancer (PCa) presents a significant health challenge in men, with a substantial number of deaths attributed to metastatic castration resistant PCa (mCRPC). Moreover, African American men experience disproportionately high mortality rates due to PCa. This study delves into the pivotal role of SPDEF, a prostate specific Ets transcription factor, and its regulation by DNA methylation in the context of PCa progression. Methods: We performed Epigenetic reprogramming using daily treatment with non-toxic dose of 5Aza-2-deoxycytidine (5Aza-dC) for two weeks to assess its impact on PDEF expression in prostate cancer cells. Next, we conducted functional studies on reprogrammed cells, including cell migration (wound-healing assay), invasion (Boyden-Chamber test), and proliferation (MTT assay) to comprehensively evaluate the consequences of altered PDEF expression. We used bisulfite sequencing (BSP) to examine DNA methylation at SPDEF promoter. Simultaneously, we utilized siRNA-mediated targeting of key DNMTs (DNMT1, DNMT3A, and DNMT3B) to elucidate their specific role in regulating PDEF. We measured mRNA and protein expressions using qRT-PCR and immune-blotting techniques, respectively. Results: In this report, we observed that: a) there is a gradual decrease in SPDEF expression with a concomitant increase in methylated CpG sites within the SPDEF gene during prostate cancer progression from lower to higher Gleason grade; b) Expression of DNMT's (DNMT1, 3a and 3b) is increased during prostate cancer progression, and there is an inverse correlation between SPDEF and DNMT expression; c) SPDEF levels are decreased in RC77/T, a line of PCa cells from African American origin similar to PC3 and DU145 cells (CRPC cells), as compared to LNCaP cells , a line of androgen dependent cells,; d) the 5' CpG island of SPDEF gene are hypermethylated in SPDEF-negative CRPC ( PC3, DU145 and RC77/T) cell lines but the same regions are hypomethylated in SPDEF-positive castrate sensitive (LNCaP) cell line ; (e) expression of SPDEF in PCa cells lacking SPDEF decreases cell migration and invasion, but has no significant effect on cell proliferation, and; (f) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, or silencing of the DNMT's by siRNA, partially restores SPDEF expression in SPDEF-negative PCa cell lines, and decreases cell migration and invasion. Discussion: These results indicate hypermethylation is a prevalent mechanism for decreasing SPDEF expression during prostate cancer progression. The data demonstrate that loss of SPDEF expression in prostate cancer cells, a critical step in cellular plasticity, results from a potentially reversible process of aberrant DNA methylation. These studies suggest DMNT activity as a potential therapeutic vulnerability that can be exploited for limiting cellular plasticity, tumor progression, and therapy resistance in prostate cancer.

Retraction notice to "Differential effects of valproic acid on growth, proliferation and metastasis in HTB-5 and HTB-9 bladder cancer cell lines" [Canc. Lett. 281/2 (2009) 196-202].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article is being retracted following correspondence from an Investigation Committee at the University of Colorado Denver. An internal investigation into this manuscript by the University of Colorado Denver, found evidence that there was image manipulation and that these actions warrant retraction to correct the scientific record. Bands on blot obscured or removed, apparent when the images are enhanced (Fig 4A p21 band "C" at 24hr, Fig 4B p21 band "C" at 24hr, and Fig 4B Cyclin A band "10" at 48 hr).

Severe COVID-19 Is Characterized by an Impaired Type I Interferon Response and Elevated Levels of Arginase Producing Granulocytic Myeloid Derived Suppressor Cells.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19 that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Hippo pathway: Regulation, deregulation and potential therapeutic targets in cancer.

Hippo pathway is a master regulator of development, cell proliferation, stem cell function, tissue regeneration, homeostasis, and organ size control. Hippo pathway relays signals from different extracellular and intracellular events to regulate cell behavior and functions. Hippo pathway is conserved from Protista to eukaryotes. Deregulation of the Hippo pathway is associated with numerous cancers. Alteration of the Hippo pathway results in cell invasion, migration, disease progression, and therapy resistance in cancers. However, the function of the various components of the mammalian Hippo pathway is yet to be elucidated in detail especially concerning tumor biology. In the present review, we focused on the Hippo pathway in different model organisms, its regulation and deregulation, and possible therapeutic targets for cancer treatment.

Pancreatic cancer: genetics, disease progression, therapeutic resistance and treatment strategies.

Pancreatic cancer is a deadly disease and the third-highest cause of cancer-related deaths in the United States. It has a very low five-year survival rate (< 5%) in the United States as well as in the world (about 9%). The current gemcitabine-based therapy soon becomes ineffective because treatment resistance and surgical resection also provides only selective benefit. Signature mutations in pancreatic cancer confer chemoresistance by deregulating the cell cycle and promoting anti-apoptotic mechanisms. The stroma-rich tumor microenvironment impairs drug delivery and promotes tumor-specific immune escape. All these factors render the current treatment incompetent and prompt an urgent need for new, improved therapy. In this review, we have discussed the genetics of pancreatic cancer and its role in tumor evolution and treatment resistance. We have also evaluated new treatment strategies for pancreatic cancer, like targeted therapy and immunotherapy.

Prostate-Derived ETS Factor (PDEF) Modulates Yes Associated Protein 1 (YAP1) in Prostate Cancer Cells: A Potential Cross-Talk between PDEF and Hippo Signaling.

PDEF (prostate-derived ETS factor, also known as SAM-pointed domain containing ETS transcription factor (SPDEF)) is expressed in luminal epithelial cells of the prostate gland and associates with luminal phenotype. The Hippo pathway regulates cell growth/proliferation, cellular homeostasis, and organ development by modulating phosphorylation of its downstream effectors. In previous studies, we observed decreased levels of PDEF during prostate cancer progression. In the present study, we evaluated the effects of the expression of PDEF on total/phosphoprotein levels of YAP1 (a downstream effector of the Hippo pathway). We observed that the PC3 and DU145 cells transfected with PDEF (PDEF-PC3 and PDEF-DU145) showed an increased phospho-YAP1 (Ser127) and total YAP1 levels as compared to the respective PC3 vector control (VC-PC3) and DU145 vector control cells (VC-DU145). We also observed an increased cytoplasmic YAP1 levels in PDEF-PC3 cells as compared to VC-PC3 cells. Moreover, our gene set enrichment analysis (GSEA) of mRNA expression in PDEF-PC3 and VC-PC3 cells revealed that PDEF resulted in inhibition of YAP1 target genes, directly demonstrating that PDEF plays a critical role in modulating YAP1 activity, and by extension in the regulation of the Hippo pathway. We also observed a decrease in YAP1 mRNA levels in prostate cancer tissues as compared to normal prostate tissues. Our analysis of multiple publicly available clinical cohorts revealed a gradual decrease in YAP1 mRNA expression during prostate cancer progression and metastasis. This decrease was similar to the decrease in PDEF levels, which we had reported earlier, and we observed a direct correlation between PDEF and YAP1 expression in CRPC data set. To the best of our knowledge, these results provide the first demonstration of inhibiting YAP1 activity by PDEF in any system and suggest a cross-talk between PDEF and the Hippo signaling pathway.

Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1): a target for cancer therapeutic intervention?

BACKGROUND: Cap-dependent mRNA translation is essential for the translation of key oncogenic proteins at optimal levels and is highly regulated by the rate limiting, initiation step in protein synthesis. Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) serves as a scaffold for assembly of cap-dependent translation components in EIF4F complex formation. In the current study, we analyzed the role and expression of EIF4G1 in Pan human cancer panels through various approaches. METHODS: Immunohistochemistry analysis of EIF4G1 protein was done on high-density multi-organ Human Cancer tissue microarray (TMA) derived from the patient samples from different cancers. We used multiple clinical cohorts to analyze the EIF4G1 mRNA expression across human cancers. TCGA data analysis of EIF4G1 was done through Ualcan and c-bioportal web servers. Western blots for EIF4G1 protein was done for different cell lines in representing the multiple cancer types. Dependency score was calculated through Cancer Dependency Map. Clonogenic, tumorosphere assay and cell invasion assay were done with EIF4G complex inhibitor. Association of EIF4G1 mRNA and Kaplan-Meier survival analysis was done on available TCGA datasets. RESULTS: We observed an increase in EIF4G1 protein levels in tissue sections from different cancers as compared to their respective normal tissue. Our analysis of the TCGA data revealed that EIF4G1 mRNA expression is significantly increased in tumor tissues compared to respective control tissues across human cancers and variable expression was observed among different datasets. We discovered that alteration frequency in EIF4G1 is prevalent in human cancers e.g. prostate cancer (~ 25%), ovarian cancer (~ 15%), Head and Neck cancer (~ 13%) and cervical cancer (~ 12.5%). EIF4G1 mRNA and protein levels were high across cancer cell lines from multiple organs. Our analysis of DepMap datasets utilizing depletion assays revealed that EIF4G1 is critical for cancer cell survival. Treatment with EIF4G complex inhibitor impaired clonogenic, tumorosphere formation potential and inhibited cell invasion. Moreover, higher EIF4G1 mRNA level was associated with a lower median survival of patients in multiple tumor types. CONCLUSIONS: These studies show that EIF4G1 is amplified/over-expressed in multiple cancers and plays an essential role in cancer cell survival, as such EIF4G1 could serve as a novel potential target for therapeutic intervention across many cancers.

Protocadherin 7 is overexpressed in castration resistant prostate cancer and promotes aberrant MEK and AKT signaling.

BACKGROUND: Castrate resistant prostate cancer (CRPC) accounts for almost all prostate cancer (PCa) deaths. Aberrant activation of ERK/MEK and PI3K/AKT signaling pathways plays an important role in subsets of patients with CRPC. The role of protocadherin 7 (PCDH7) in modulating these signaling pathways is investigated for the first time in PCa in the present investigation. METHODS: PCDH7 expression was analyzed in CRPC/neuroendocrine prostate cancer (NEPC) dataset. Protein expression was assessed by Western blotting and immunohistochemistry, and messenger RNA (mRNA) by quantitative real-time polymerase chain reaction. Small hairpin ribonucleic acid was used to knockdown PCDH7. Colony formation, cell migration, and invasion studies were done using standard protocols. RESULTS: PCDH7 amplification/mRNA upregulation was observed in 41% of patients in CRPC/NEPC dataset. PCDH7 was also overexpressed in CRPC cells. Increased PCDH protein expression was observed during tumor progression in PCa tissues and in TRAMP mice. Epidermal growth factor treatment resulted in aberrant activation of ERK/AKT. Knockdown of PCDH7 decreased ERK, AKT, and RB phosphorylation and reduced colony formation, decreased cell invasion, and cell migration. CONCLUSIONS: These data show for the first time that PCDH7 is overexpressed in a large number of patients with CRPC and suggest that PCDH7 may be an attractive target in subsets of patients with CRPC for whom there is no cure to-date.

The TLK1-Nek1 axis promotes prostate cancer progression.

We recently uncovered the critical TLK1>NEK1>ATR > Chk1 axis in mediating the DDR and cell cycle checkpoint while transiting from Androgen Sensitive to Insensitive growth for LNCaP and TRAMP-C2 cells. However, we did not know the generality of this pathway in PCa progression since there are few cell lines where the transition has been studied. Furthermore, the identification of Nek1, and more importantly the TLK-mediated phosphorylation of T141, has never been studied in PCa biopsies. We now report the first study of a PCa TMA of p-Nek1-T141 and correlation to the Gleason score. In addition we found that TRAMP mice treated with the TLK inhibitor, thioridazine (THD), following castration did not recover cancerous growth of their prostates. Moreover, we recapitulated the process of translational increase in TLK1B expression in a naïve PDX model that was established from an AR + adenocarcinoma. Therefore, we believe that this TLK1-Nek1 mediated DDR axis is likely to be a common adaptive response during the transition of PCa cells toward androgen-insensitive growth, and hence CRPC progression, which has the potential to be targeted with THD and other TLK or Nek1 inhibitors.

Reactive oxygen species and cancer: A complex interaction.

Elevated levels of Reactive Oxygen Species (ROS), increased antioxidant ability and the maintenance of redox homeostasis can cumulatively contribute to tumor progression and metastasis. The sources and the role of ROS in a heterogeneous tumor microenvironment can vary at different stages of tumor: initiation, development, and progression, thus making it a complex subject. In this review, we have summarized the sources of ROS generation in cancer cells, its role in the tumor microenvironment, the possible functions of ROS and its important scavenger systems in tumor progression with special emphasis on solid tumors.

Targeting the TLK1/NEK1 DDR axis with Thioridazine suppresses outgrowth of androgen independent prostate tumors.

Standard therapy for advanced Prostate Cancer (PCa) consists of antiandrogens, which provide respite from disease progression, but ultimately fail resulting in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would greatly improve the outcome. Combination therapy targeting the DNA Damage Response (DDR) has been limited by general toxicity, and a goal of clinical trials is how to target the DDR more specifically. We now show that androgen deprivation therapy (ADT) of LNCaP cells results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DDR that typically results in a temporary cell cycle arrest of androgen responsive PCa cells. Following DNA damage, addition of the TLK specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of a ADT > TLK1 > NEK1 > ATR > Chk1, DDR pathway, while its abrogation leads to apoptosis. Treatment with THD suppressed the outgrowth of androgen-independent (AI) colonies of LNCaP and TRAMP-C2 cells cultured with bicalutamide. Moreover, THD significantly inhibited the growth of several PCa cells in vitro (including AI lines). Administration of THD or bicalutamide was not effective at inhibiting long-term tumor growth of LNCaP xenografts. In contrast, combination therapy remarkably inhibited tumor growth via bypass of the DDR. Moreover, xenografts of LNCaP cells overexpressing a NEK1-T141A mutant were durably suppressed with bicalutamide. Collectively, these results suggest that targeting the TLK1/NEK1 axis might be a novel therapy for PCa in combination with standard of care (ADT).

Prostate-Derived Ets Factor (PDEF) Inhibits Metastasis by Inducing Epithelial/Luminal Phenotype in Prostate Cancer Cells.

Metastasis is the primary cause of prostate cancer morbidity and mortality. Our previous studies revealed that Sam pointed domain ETS transcription factor, a.k.a. prostate-derived ETS factor (SPDEF/PDEF), inhibits prostate cancer metastasis. However, the mechanism is still unclear. In this study, using microarray and gene set enrichment analysis, we discovered that PDEF upregulated epithelial/luminal differentiation-related genes while it suppressed stemness and epithelial-to-mesenchymal transition-related genes, especially Twist1. We also observed loss of PDEF and gain of Twist1 expression during prostate cancer progression in the TRAMP mouse model. Moreover, Twist1 knockdown resulted in upregulation of PDEF expression, suggesting a reciprocal regulation between PDEF and Twist1. Mechanistically, our ChIP-seq analysis revealed that PDEF directly regulated cytokeratin 18 (CK18) transcription through the GGAT motif within its putative promoter region. CK18 knockdown resulted in increased expression of Twist1, suggesting that PDEF regulated Twist1 in part via CK18. Our analysis of multiple clinical prostate cancer cohorts revealed an inverse relationship between PDEF expression and tumor grade, tumor metastasis, and poor patient survival. Furthermore, a two-gene signature of low PDEF and high Twist1 can better predict poor survival in prostate cancer patients than either gene alone. Collectively, our findings demonstrate PDEF inhibits prostate tumor progression, in part, by directly regulating transcription of CK18, and that PDEF/Twist1 expression could help distinguish between lethal and indolent prostate cancer.Implications: This study reports the novel findings that PDEF suppresses Twist1 partly via CK18 and that PDEF/Twist1 could help distinguish between lethal and indolent prostate cancer.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/9/1430/F1.large.jpg Mol Cancer Res; 16(9); 1430-40. ©2018 AACR.

Eukaryotic Translation Initiation Factor 4 Gamma 1 (eIF4G1) is upregulated during Prostate cancer progression and modulates cell growth and metastasis.

eIF4G1, a critical component of the eIF4F complex, is required for cap-dependent mRNA translation, a process necessary for tumor growth and survival. However, the role of eIF4G1 has not been evaluated in Prostate Cancer (PCa). We observed an increased eIF4G1 protein levels in PCa tissues as compared to normal tissues. Analysis of the TCGA data revealed that eIF4G1 gene expression positively correlated with higher tumor grade and stage. Furthermore, eIF4G1 was over-expressed and or amplified, in 16% patients with metastatic PCa (SU2C/PCF Dream Team dataset) and in 59% of castration-resistant prostate cancer (CRPC) patients (Trento/Cornell/Broad dataset). We showed for the first time that eIF4G1 expression was increased in PCa and that increased eIF4G1 expression associated with tumor progression and metastasis. We also observed high protein levels of eIF4G1 in PCa cell lines and prostate tissues from the TRAMP model of PCa as compared to normal prostate cell line and prostate tissues from the wild type mice. Knockdown of eIF4G1 in PCa cells resulted in decreased Cyclin D1 and p-Rb protein level, cell cycle delay, reduced cell viability and proliferation, impaired clonogenic activity, reduced cell migration and decreased mRNA loading to polysomes. Treatment with eIF4G complex inhibitor also impaired prostasphere formation. eIF4G1 knockdown or treatment with eIF4G complex inhibitor sensitized CRPC cells to Enzalutamide and Bicalutamide. Our results showed that eIF4G1 plays an important role in PCa growth and therapeutic resistance. These data suggested that eIF4G1 functions as an oncoprotein and may serve as a novel target for intervention in PCa and CRPC.

Tetrandrine inhibits deregulated cell cycle in pancreatic cancer cells: Differential regulation of p21Cip1/Waf1, p27Kip1 and cyclin D1.

Current therapies in Pancreatic Cancer (PaCa) are ineffective due to deregulated cell cycle driven by landscape mutations. In this study, we show for the first time that tetrandrine (TET) inhibits proliferation of the PaCa cells and inhibits PaCa tumor growth. TET inhibits cell cycle transition at G1/S boundary. TET increased levels of p21Cip1/Waf1 and p27Kip1, had no effect on the levels of CDK4/6 proteins and decreased the levels of cyclin D1 and pRb proteins. TET resulted in changes in mRNA levels of cyclin D1 and p21Cip1/Waf1 but had no effect on the mRNA of p27Kip1. We show, for the first time in any system, that TET treatment downregulated Skp2, E3 ligase specific for degradation of p27Kip1 during the cell cycle. Taken together our results show, that TET indirectly impairs the activities of CDK4/6 to halt deregulated cell cycle and inhibit PaCa tumor growth. These results suggest that TET may serve as a novel agent for treatment of PaCa, for which there is no effective cure to date.

Tetrandrine (TET) Induces Death Receptors Apo Trail R1 (DR4) and Apo Trail R2 (DR5) and Sensitizes Prostate Cancer Cells to TRAIL-Induced Apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells, but not in normal cells; as such, it is a promising therapeutic agent. However, therapeutic resistance limits its clinical use in many malignancies, including prostate cancer. Strategies to sensitize cancer cells to TRAIL are urgently needed. We demonstrate here that small-molecule tetrandrine (TET) potentially sensitizes previously resistant (LNCaP and C4-2B cells) and mildly sensitive (PC3 cells) prostate cancer cells to TRAIL-induced apoptosis, and they do so by upregulating mRNA expression and protein levels of death receptors Apo Trail R1 (DR4) and Apo Trail R2 (DR5). Using shRNA knockdown, we show critical requirement of DR4 and DR5 in sensitization of prostate cancer cells to TRAIL. We show that double knockdown of DR4 and DR5 abrogated the apoptotic effects of TET and TRAIL. We also demonstrate that TET-induced DR4 and DR5 expression is independent of p53 status. Given that loss of p53 is associated with progression of prostate cancer to CRPC and NEPC, our results show that TET, by acting as a TRAIL-sensitizing agent in prostate cancer, could serve as a potential therapeutic agent in CRPC and NEPC, for which there is no cure to date. Mol Cancer Ther; 17(6); 1217-28. ©2018 AACR.