Prostate Cancer Cell Lines 22Rv1 Research Articles

Pinostilbene inhibits full-length and splice variant of androgen receptor in prostate cancer

Prostate cancer is the most prevalent cancer in men worldwide and is promoted by the sex hormone androgen. Expression of androgen from the testis can be significantly reduced through castration. However, as most prostate cancer patients acquire castration resistance, additional therapeutic solutions are necessary. Although anti-androgens, such as enzalutamide, have been used to treat castration-resistant prostate cancer (CRPC), enzalutamide-resistant CRPC (Enz-resistant CRPC) has emerged. Therefore, development of novel treatments for Enz-resistant CRPC is urgent. In this study, we found a novel anti-androgen called pinostilbene through screening with a GAL4-transactivation assay. We confirmed that pinostilbene directly binds to androgen receptor (AR) and inhibits its activation and translocalization. Pinostilbene treatment also reduced the protein level and downstream gene expression of AR. Furthermore, pinostilbene reduced the protein level of AR variant 7 in the Enz-resistant prostate cancer cell line 22Rv1 and inhibited cell viability and proliferation. Our results suggest that pinostilbene has the potential to treat Enz-resistant CRPC.

The Impact of TP53 Mutations and Use of the TP53-Mutation-Reactivating Agent APR-246 on Metastatic Castrate-Sensitive Prostate Cancer.

TP53 mutations appear to be enriched over the spectrum of metastatic castration-sensitive prostate cancer (mCSPC) and are associated with worse survival outcomes. We chose to further explore the impact of dominant negative (DN) TP53 mutations on mCSPC progression and pro-metastatic behaviors in addition to studying the ability of APR-246, a small molecule targeting TP53 mutants, to blunt pro-metastatic behaviors. We retrospectively analyzed 531 mCSPC patients who underwent next-generation sequencing. Patients were stratified by metastasis timing (synchronous if metastasis present at diagnosis or metachronous if arising after definitive treatment of localized disease) and the number of metastatic lesions (oligometastatic ≤5 or polymetastatic >5 lesions). Tumors were classified based on TP53 mutation status (missense, truncating, or wild-type [WT]) and dominant negativity, which was defined as the production of a mutant protein that reduces the residual WT protein's transcriptional activity according to the World Health Organization TP53 database. Clinical outcomes were radiographic progression-free survival (rPFS) and overall survival (OS), evaluated with Kaplan-Meier and multivariable Cox regression. To verify the impact of TP53 mutation on metastasis, we created isogenic 22Rv1 prostate cancer cell lines that carried either TP53 WT or TP53 R175H and tested this mutation for migration, invasion, and anchorage-independent growth. APR-246 (25-80 µM) was tested for anti-metastatic properties in vitro and anti-tumor growth in 22Rv1 xenografted nude mice. In our cohort, 155 (29.2%) had a TP53 mutation, which mostly occurred in the DNA-binding domain (85.16%). DN TP53 mutations were associated with more aggressive disease states: DN TP53 mutations were enriched in patients with synchronous (vs. metachronous: 20.7% vs. 6.3%, p < 0.01) and polymetastatic disease (vs. oligometastatic: 14.4% vs. 7.9%, p < 0.01). On multivariable analysis, DN TP53 mutations were correlated with shorter rPFS (HR = 1.97, 95% CI: 1.31-2.98, p < 0.01) and OS (HR = 2.05, 95% CI: 1.14-3.68, p = 0.02) compared to those with TP53 WT. In vitro, 22Rv1 cells with DN TP53 R175H mutation had increased abilities to migrate, invade, and form colonies compared to TP53 WT. APR-246 treatment of TP53 R175H mutants blunted the pro-metastatic effects of the cell line in vitro (p < 0.01 for all assays by unpaired t-test). Interestingly, APR-246 also inhibited xenograft tumor growth of 22Rv1 TP53 R175H mutants (p < 0.0001 by two-way ANOVA). DN TP53 mutations were associated with poorer survival outcomes for mCSPC patients. DN TP53 mutations also promoted prostate cancer pro-metastatic behaviors in vitro, which was effectively counteracted by APR-246, making it a promising treatment option that should be explored further in early-phase clinical studies.

Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK/ELK1 signalling pathway.

Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.

Abstract A007: Modulation of HER2 as a mechanism for AR expression

Abstract Background: Intense neoadjuvant androgen deprivation therapy (inADT) is an emerging treatment paradigm for localized high-risk prostate cancer, but in certain patients a subset of tumors will recur after surgery. In tumors with endogenously low androgen receptor (AR) activity, expression of the EGFR family protein HER2 was enriched. We sought to investigate the role of HER2 in driving the proliferation of prostate cancer under androgen-depleted environments. We hypothesized that overexpression of HER2 would decrease AR activity, while its knockdown would conversely increase AR expression beyond baseline levels. Methods: The AR-expressing 22Rv1 and LNCaP prostate cancer cell lines were cultured and plated for transfection. HER2 overexpression was achieved using an exogenous HER2 (ERBB2) plasmid, while HER2 knockdown was performed using siRNA, alongside controls for both groups. Cell lysates were collected at 24-, 48-, and 72-hour time points. Western blotting was performed to examine HER2, AR, PSA, and actin expression. Results: Western blot analysis confirmed successful overexpression and knockdown of HER2, using an exogenous HER2 plasmid and siRNA, respectively. In 22Rv1 and LNCaP cells overexpressing HER2, AR (and AR-V7 in 22Rv1 cells) levels were decreased compared to the control cells. Conversely, 22Rv1 and LNCaP cells with knockdown of HER2 exhibited elevated AR expression. Biological replicates to confirm these findings are in progress. Additional studies will also examine the impact of HER2 signaling and activity on downstream pro-proliferative pathways. Conclusions: Manipulation of HER2 expression highlights a potential role of EGFR family proteins as a mechanism for prostate cancer growth in androgen-deprived environments. Future studies will validate this finding at the RNA level using qPCR and probe downstream targets of HER2 using western blotting. These preliminary findings have implications for the treatment of advanced tumors for which inADT is not a viable option. Targeting HER2 and its downstream signaling pathways represent an intriguing mechanism to overcome resistance to androgen deprivation therapy in newly-diagnosed, treatment-naïve patients. Citation Format: Daniel K.Y. Low, Scott Wilkinson, Isaiah M. King, Shana Y. Trostel, Adam G. Sowalsky. Modulation of HER2 as a mechanism for AR expression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A007.

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

The impact of TP53 mutations and use of the TP53-mutation-reactivating agent APR-246 on metastatic castrate-sensitive prostate cancer.

e17077 Background: TP53 mutations appear to be enriched over the spectrum of metastatic castration-sensitive prostate cancer (mCSPC) and are associated with worse survival outcomes. We chose to further explore the impact of dominant negative (DN) TP53 mutations on mCSPC progression and pro-metastatic behaviors in addition to studying the ability of APR-246, a small molecule targeting TP53 mutants, to blunt pro-metastatic behaviors. Methods: We retrospectively analyzed 531 mCSPC patients who underwent next generation sequencing. Patients were stratified by metastasis timing (synchronous if metastasis present at diagnosis or metachronous if arising after definitive treatment of localized disease) and the number of metastatic lesions (oligometastatic &lt;5 or polymetastatic &gt; 5 lesions). Tumors were classified based on TP53 mutation status (missense, truncating, or wild-type [WT]) and dominant negativity, which was defined as production of a mutant protein that reduces the residual WT protein's transcriptional activity according to World Health Organization TP53 database. Clinical outcomes were radiographic progression-free survival (rPFS) and overall survival (OS), evaluated with Kaplan-Meier and multivariable Cox regression. To verify the impact of TP53 mutation on metastasis, we created isogenic 22Rv1 prostate cancer cell lines that carried either TP53 WT or TP53 R175H and tested this mutation for migration, invasion, and anchorage-independent growth. APR-246 (25-80 µM) was tested for anti-metastatic properties in vitro and anti-tumor growth in 22Rv1 xenografted nude mice. Results: In our cohort, 155 (29.2%) had a TP53 mutation, which mostly occurred in the DNA-binding domain (85.16%). DN TP53 mutations were associated with more aggressive disease states: DN TP53 mutations were enriched in patients with synchronous (vs. metachronous: 20.7% vs. 6.3%, p &lt; 0.01) and polymetastatic disease (vs. oligometastatic: 14.4% vs. 7.9%, p &lt; 0.01). On multivariable analysis, DN TP53 mutations were correlated with shorter rPFS (HR = 1.97, 95%CI: 1.31-2.98, p &lt; 0.01) and OS (HR = 2.05, 95%CI: 1.14-3.68, p = 0.02) compared to those with TP53 WT. In vitro, 22Rv1 cells with DN TP53 R175H mutations had increased ability to migrate, invade, and form colonies compared to TP53 WT cells. APR-246 treatment of TP53 R175H mutants blunted the pro-metastatic effects of the cell line in vitro (p &lt; 0.01 for all assays by unpaired t-test). Interestingly, APR-246 inhibited xenograft tumor growth of 22Rv1 TP53 R175H prostate cancer cells (p &lt; 0.0001 by two-way ANOVA). Conclusions: DN TP53 mutations were associated with poorer survival outcomes for mCSPC patients. DN TP53 mutations also promoted prostate cancer pro-metastatic behaviors in vitro, which was effectively counteracted by APR-246, making it a promising treatment option that should be explored further in early-phase clinical studies.

One-Minute Iodine Isotope Labeling Technology Enables Noninvasive Tracking and Quantification of Extracellular Vesicles in Tumor Lesions and Intact Animals.

Real-time monitoring of the biological behavior of extracellular vesicles (EVs) in vivo is limited, which hinders its application in biomedicine and clinical translation. A noninvasive imaging strategy could provide us with useful information on EVs' distribution, accumulation and homing in vivo, and pharmacokinetics. In this study, the long half-life radionuclide iodine-124 (124I) was used to directly label umbilical cord mesenchymal stem cell-derived EVs. The resulting probe, namely, 124I-MSC-EVs, was manufactured and ready to use within 1 min. 124I-labeled MSC-EVs had high radiochemical purity (RCP, >99.4%) and stable in 5% human serum album (HSA) with RCP > 95% for 96 h. We demonstrated efficient intracellular internalization of 124I-MSC-EVs in two prostate cancer cell lines (22RV1 and DU145 cell). The uptake rates of 124I-MSC-EVs in human prostate cancer cell lines 22RV1 and DU145 cells were 10.35 ± 0.78 and 2.56 ± 0.21 (AD%) at 4 h. The promising cellular data has prompted us to investigate the biodistribution and in vivo tracking capability of this isotope-based labeling technique in tumor bearing animals. Using positron emission tomography (PET) technology, we showed that the signal from intravenously injected 124I-MSC-EVs mainly accumulated in the heart, liver, spleen, lung, and kidney in healthy kun ming (KM) mice, and the biodistribution study was similar to the imaging results. In the 22RV1 xenograft model, 124I-MSC-EVs accumulated significantly in the tumor after administration, and with the optimal image acquired at 48 h postinjection, the maximum of standard uptake value (SUVmax) of the tumor was 3-fold higher than that of DU145. Taken together, the probe has a high application prospect in immuno-PET imaging of EVs. Our technique provides a powerful and convenient tool for understanding the biological behavior and pharmacokinetic characteristics of EVs in vivo and facilitates the acquirement of comprehensive and objective data for future clinical studies of EVs.

Downstream of kinase 3 promotes proliferation and inhibits apoptosis of prostate cancer via the NF-κB signaling pathway.

Downstream of kinase 3 (DOK3) is involved primarily with immune cell infiltration. Recent research reported the role of DOK3 in tumor progression, with opposite effects in lung cancer and gliomas; however, its role in prostate cancer (PCa) remains elusive. This study aimed to explore the role of DOK3 in PCa and to determine the mechanisms involved. To investigate the functions and mechanisms of DOK3 in PCa, we performed bioinformatic and biofunctional analyses. Samples from patients with PCa were collected from West China Hospital, and 46 were selected for the final correlation analysis. A lentivirus-based short hairpin ribonucleic acid (shRNA) carrier was established for silencing DOK3. A series of experiments involving the cell counting kit-8, bromodeoxyuridine, and flow cytometry assays were performed to identify cell proliferation and apoptosis. Changes in biomarkers from the nuclear factor kappa B (NF-κB) signaling pathway were detected to verify the relationship between DOK3 and the NF-κB pathway. A subcutaneous xenograft mouse model was performed to examine phenotypes after knocking down DOK3 in vivo. Rescue experiments with DOK3 knockdown and NF-κB pathway activation were designed to verify regulating effects. DOK3 was up-regulated in PCa cell lines and tissues. In addition, a high level of DOK3 was predictive of higher pathological stages and worse prognoses. Similar results were observed with PCa patient samples. After silencing DOK3 in PCa cell lines 22RV1 and PC3, cell proliferation was significantly inhibited while apoptosis was promoted. Gene set enrichment analysis revealed that DOK3 function was enriched in the NF-κB pathway. Mechanism experiments determined that knockdown of DOK3 suppressed activation of the NF-κB pathway, increased the expressions of B-cell lymphoma-2 like 11 (BIM) and B-cell lymphoma-2 associated X (BAX), and decreased the expression of phosphorylated-P65 and X-linked inhibitor of apoptosis (XIAP). In the rescue experiments, pharmacological activation of NF-κB by tumor necrosis factor-α (TNF-α) partially recovered cell proliferation after the knockdown of DOK3. Our findings suggest that overexpression of DOK3 promotes PCa progression by activating the NF-κB signaling pathway.

Transcripts of the Prostate Cancer-Associated Gene ANO7 Are Retained in the Nuclei of Prostatic Epithelial Cells.

Prostate cancer affects millions of men globally. The prostate cancer-associated gene ANO7 is downregulated in advanced prostate cancer, whereas benign tissue and low-grade cancer display varying expression levels. In this study, we assess the spatial correlation between ANO7 mRNA and protein using fluorescent in situ hybridization and immunohistochemistry for the detection of mRNA and protein in parallel sections of tissue microarrays prepared from radical prostatectomy samples. We show that ANO7 mRNA and protein expression correlate in prostate tissue. Furthermore, we show that ANO7 mRNA is enriched in the nuclei of the luminal cells at 89% in benign ducts and low-grade cancer, and at 78% in high-grade cancer. The nuclear enrichment of ANO7 mRNA was validated in prostate cancer cell lines 22Rv1 and MDA PCa 2b using droplet digital polymerase chain reaction (ddPCR) on RNA isolated from nuclear and cytoplasmic fractions of the cells. The nuclear enrichment of ANO7 mRNA was compared to the nuclearly-enriched lncRNA MALAT1, confirming the surprisingly high nuclear retention of ANO7 mRNA. ANO7 has been suggested to be used as a diagnostic marker and a target for immunotherapy, but a full comprehension of its role in prostate cancer progression is currently lacking. Our results contribute to a better understanding of the dynamics of ANO7 expression in prostatic tissue.

Ginsenoside compound K suppresses tumour growth in the 22Rv1 xenograft model and inhibits androgenic responses via the transcriptional mechanism in human prostate cancer cells.

Androgen receptor (AR) signalling is known to be dispensable for the biology of castration-resistant prostate cancer (CRPC), whereas the AR itself and the residual androgens after castration are crucial for the growth and progression of CRPC. Therefore, there is high demand for novel therapeutic candidates targeting AR itself or aberrant AR signalling to suppress the progression to or the growth of CPRC. Here, we report that ginsenoside compound K (GCK), the primary bioactive metabolite biotransformed from protopanaxadiol (PPD) ginsenoside, acts as a novel AR signalling inhibitor by transcriptionally suppressing AR expression and tumour growth in athymic nude mice. GCK inhibited cell growth in LNCaP, PC-3 and 22Rv1 prostate cancer cell lines and suppressed the expression levels of cell cycle regulators. GCK down-regulated epithelial-mesenchymal transition markers such as vimentin and matrix metalloproteinase 9 (MMP9), whereas E-cadherin was significantly increased in GCK-stimulated LNCaP and 22Rv1 cells. Moreover, GCK treatment markedly decreased both AR and AR-V7 protein levels in LNCaP and 22Rv1 cells, possibly by decreasing AR promoter activity. Experiments with AR promoter-deleted constructs revealed that the region between -412 and -227 is critical for GCK regulation. GCK treatment in athymic nude mice implanted with 22Rv1 CRPC cell lines significantly suppressed tumour growth and AR expression levels in tumour tissues. Collectively, our results suggest that GCK, as a novel AR inhibitor, could be a potential therapeutic agent against prostate cancer and an effective chemopreventive agent to delay the progression to CRPC.

306 (PB086) - Reversal of resistance to docetaxel and cabazitaxel in prostate cancer cells with ritonavir

Background: The taxanes docetaxel and cabazitaxel are indicated for treatment of metastatic castration-resistant prostate cancer (mCRPC). Resistance to docetaxel often develops during treatment, which can be due to an increased efflux of docetaxel via P-glycoprotein (P-gp) from the intracellular site of action. Ritonavir is an inhibitor of both cytochrome P4503A4 (CYP3A4) and P-gp. In this study we investigated whether ritonavir could improve the efficacy of docetaxel through inhibition of P-gp in docetaxel-resistant prostate cancer cells. Methods: Prostate cancer cell lines DU-145 and 22Rv1 were exposed to increasing doses of docetaxel to generate resistant sublines, DU145-DOC10 and 22Rv1DOC8 respectively. Gene expression of the resistant clones was compared to that of the parental cell lines. The parental cell lines and the docetaxel resistant cell lines were treated with several drug combinations in an 8*2 matrix 3D tumor clonogenic assay. The following drug combinations were tested: docetaxel/ritonavir, docetaxel/elacridar and cabazitaxel/ritonavir. The IC50 values of single agent drugs and drug combinations as well as combination effects between drugs (using a Bliss independence dose-response evaluation) were determined in all cell lines. Results: The most differentially upregulated gene in the resistant cell lines was ABCB1 (P-gp). Western blotting showed that both resistant cell lines expressed more P-gp than the parental cell lines. A comparison of the observed docetaxel and cabazitaxel IC50 values has been summarized in the below table.Table(abstract: 306 (PB086))DU-145DU-145DOC10DU-145DOC1022Rv122Rv1DOC822Rv1DOC8Docetaxel IC50 (nM)2.99.52.8*with 10 μM ritonavir,1.354.63.0**with 32 μM ritonavir.Cabazitaxel IC50 (nM)0.61.50.6*with 10 μM ritonavir,0.79.50.7**with 32 μM ritonavir.* with 10 μM ritonavir,** with 32 μM ritonavir. Open table in a new tab We found that docetaxel resistance conferred cross-resistance to cabazitaxel. With the addition of 10 μM ritonavir (DU-145DOC10) and 32 μM ritonavir (22Rv1DOC8) the cytoxicity of docetaxel and cabazitaxel returned to the level of observed in the parental cell lines. A similar experiment with the specific P-gp inhibitor elacridar showed that a concentration of 0.3 μM was sufficient to reverse docetaxel resistance to the same level as in the parental cell lines, confirming this as an important mechanism-of-action. In DU-145-DOC10 and 22Rv1DOC8 cells strong synergistic effects were seen of the docetaxel/ritonavir as well as the cabazitaxel/ritonavir combination, in contrast to the lack of synergy seen in the parental cell lines with these combinations. Conclusions: We demonstrated the potential for the combination of docetaxel and ritonavir to reverse or offset resistance to docetaxel (and, by implication due to its cross-resistance, also to cabazitaxel), primarily mediated through inhibition of P-gp. This provides a strong rationale to clinically test these taxanes in combination with ritonavir in patients with mCRPC. Conflict of interest: Ownership: Jos Beijnen is a shareholder of Modra Pharmaceuticals. Board of Directors: Colin Freund and Eric van der Putten are directors of Modra Pharmaceuticals.

SIRT4 functions as a tumor suppressor during prostate cancer by inducing apoptosis and inhibiting glutamine metabolism

Localized in the mitochondria, SIRT4 is a nicotinamide adenine dinucleotide (NAD +) -dependent adenosine diphosphate (ADP) -ribosyltransferase and is one of the least characterized members of the sirtuin family. Although it is well known that it shows deacetylase activity for energy metabolism, little is understood about its function in tumorigenesis. Recent research suggests that SIRT4 may work as both a tumor suppressor gene and an oncogene. However, the clinical significance of SIRT4 in prostate cancer remains unknown. In this study, we evaluated SIRT4 protein levels in cancerous prostate tissue and corresponding non-tumor prostate tissue via immunohistochemical staining on a tissue microarray including tissues from 89 prostate cancer patients. The association between SIRT4 expression and Gleason score was also determined. Further, shSIRT4 or stable prostate cancer cell lines (22RV1) overexpressing SIRT4 were constructed via lentiviral infection. Using Cell-Counting Kit-8 (CCK-8) assay, wound healing assay, migration, and invasion and apoptosis assays, the effects of SIRT4 on the migration, invasion ability, and proliferation of prostate cancer cells were investigated. We also determined the effect of SIRT4 on glutamine metabolism in 22RV1 cells. We found the protein levels of SIRT4 in prostate cancer tissues were significantly lower than those in their non-neoplastic tissue counterparts (P < 0.01); a lower SIRT4 level was also significantly associated with a higher Gleason score (P < 0.01). SIRT4 suppressed the migration, invasion capabilities, and proliferation of prostate cancer cells and induced cellular apoptosis. Furthermore, the invasion and migration of 22RV1 cells were mechanistically inhibited by SIRT4 via glutamine metabolism inhibition. In conclusion, the present study’s findings showed that SIRT4 protein levels are significantly associated with the Gleason score in patients with prostate cancer, and SIRT4 exerts a tumor-suppressive effect on prostate cancer cells by inhibiting glutamine metabolism. Thus, SIRT4 may serve as a potential novel therapeutic target for prostate cancer.

Abstract 4096: Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members

Abstract Ubiquitination is a metabolic process in eukaryotic cells that modifies proteins with ubiquitin leading to protein degradation via the ubiquitin-proteosome pathway. Cellular levels of ubiquitin are driven by the expression of RPS27A, UBA52, UBB and UBC. In cancer, the dysregulation of ubiquitination can affect various aspects of tumor progression including cell cycle regulation and gene expression. Ubiquitin B (UBB) gene has been shown to be over-expressed in some tumor types, including NSCLC and cervical cancer, and under-expressed in other tumors, in particularly a subset of ovarian cancers. A role for ubiquitin C (UBC) in cancer has not been well established but UBC has been described as a biomarker in renal cancer and CLL. In recent studies, repression of UBB in ovarian cancer cells was associated with sensitivity to UBC knockdown and demonstrated a synthetic lethal relationship. Since both UBB and UBC show substantial sequence homology, we explored the possibility of developing dual targeting siRNA that would silence both UBB and UBC. We identified unique individual siRNA sequences that efficiently silenced both UBB and UBC and led to rapid cytotoxicity in several cancer cell lines including the colon cancer cell line HCT-116, the breast cancer cell line SK-BR3, and the prostate cancer cell line 22Rv1. Treatment with either UBB-specific or UBC-specific siRNA did not show the same lethal phenotype. Dose response assays using the dual targeting siRNA designated U21 indicated EC50 for cytotoxicity between 50–500 pM in several cancer lines. Treatment of cancer cell lines with U21 leads to rapid apoptosis and cell death. Chemical modifications to pharmacologically stabilize the U21 siRNA sequence did not change its activity. The efficient cytotoxic activity of the U21 siRNA sequence has been integrated into our SeekR™ platform to create aptamer-siRNA chimeras for targeted delivery of the lethal U21 payload to specific cancer cell types. Citation Format: David O. Azorsa, David W. Lee, Marvin O'Ketch, Nathalie A. Azorsa, Ioanna Papacharalampous, Lizette A. Castaño, Jeffrey A. Kiefer, Spyro Mousses. Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4096.

TNFRSF13B is a potential contributor to prostate cancer.

BackgroundImmunodeficiencies are genetic diseases known to predispose an individual to cancer owing to defective immunity towards malignant cells. However, the link between immunodeficiency and prostate cancer progression remains unclear. Therefore, the aim of this study was to evaluate the effects of common genetic variants among eight immunodeficiency pathway-related genes on disease recurrence in prostate cancer patients treated with radical prostatectomy.MethodsGenetic and bioinformatic analyses on 19 haplotype-tagging single-nucleotide polymorphisms in eight immunodeficiency pathway-related genes were conducted in 458 patients with prostate cancer after receiving radical prostatectomy. Furthermore, the TNFRSF13B was knocked down in 22Rv1 and PC-3 human prostate cancer cell lines via transfecting short hairpin RNAs and cell proliferation and colony formation assays were performed. The molecular mechanisms underlying the effects of TNFRSF13B were further explored by microarray gene expression profiling.ResultsTNFRSF13B rs4792800 was found to be significantly associated with biochemical recurrence even after adjustment for clinical predictors and false discovery rate correction (adjusted hazard ratio 1.78, 95% confidence interval 1.16–2.71, p = 0.008), and the G allele was associated with higher TNFRSF13B expression (p = 0.038). Increased TNFRSF13B expression suggested poor prognosis in four independent prostate cancer datasets. Furthermore, silencing TNFRSF13B expression resulted in decreased colony formation of 22Rv1 and PC-3 cells through modulating the cell cycle and p53 signalling pathways.ConclusionsThe present study suggests the potential role of immunodeficiency pathway-related genes, primarily TNFRSF13B, in prostate cancer progression.Supplementary informationThe online version contains supplementary material available at 10.1186/s12935-022-02590-2.

Thiodiketopiperazines and Alkane Derivatives Produced by the Mangrove Sediment–Derived Fungus Penicillium ludwigii SCSIO 41408

A new trithiodiketopiperazine derivative, adametizine C (1), and five new alkane derivatives (7–11), were isolated from the mangrove sediment–derived fungus Penicillium ludwigii SCSIO 41408, together with five known dithiodiketopiperazine derivatives (2–6). Their structures were elucidated on the basis of spectroscopic analysis, and the absolute configuration of 1 was determined by X-ray crystallographic analysis. In a variety of bioactivity screening, 1–5 exhibited some selective antifungal or antibacterial activities. Compounds 1–3 showed cytotoxicity against prostate cancer cell line 22Rv1 with half maximal inhibitory concentration (IC50) values of 13.0–13.9 μM; moreover, 3 showed obvious activity against another prostate cancer PC-3 cells with an IC50 value of 5.1 μM. Further experiments revealed that 3 could significantly reduce PC-3 cells colony formation and induce apoptosis in a dose-dependent manner. Several compounds also exhibited obvious inhibitory activities of lipopolysaccharide–induced nuclear factor-κB with IC50 values range from 8.2 to 21.5 μM, and 1, 5, and 9 were further evaluated for their effects on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. Adametizine C (1), with the strongest inhibitory activity against RANKL-induced osteoclast differentiation in bone marrow macrophage cells with 10 μM, was suggested to be the promising lead compound for the treatment of osteoclast-related diseases.

Indolethylamine-N-Methyltransferase Inhibits Proliferation and Promotes Apoptosis of Human Prostate Cancer Cells: A Mechanistic Exploration.

Indolethylamine-N-methyltransferase (INMT) is a methyltransferase downregulated in lung cancer, meningioma, and prostate cancer; however, its role and mechanism in prostate cancer remain unclear. By analyzing The Cancer Genome Atlas (TCGA)-PRAD, we found that the expression of INMT in prostate cancer was lower than that of adjacent non-cancerous prostate tissues and was significantly correlated with lymph node metastasis Gleason score, PSA expression, and survival. Combined with the GSE46602 cohorts for pathway enrichment analysis, we found that INMT was involved in regulating the MAPK, TGFβ, and Wnt signaling pathways. After overexpression of INMT in prostate cancer cell lines 22Rv1 and PC-3, we found an effect of INMT on these tumor signal pathways; overexpression of INMT inhibited the proliferation of prostate cancer cells and promoted apoptosis. Using the ESTIMATE algorithm, we found that with the increase of INMT expression, immune and stromal scores in the tumor microenvironment increased, immune response intensity increased, and tumor purity decreased. The difference in INMT expression affected the proportion of several immune cells. According to PRISM and CTRP2.0, the potential therapeutic agents associated with the INMT expression subgroup in TCGA were predicted. The area under the curve (AUC) values of 26 compounds positively correlated with the expression of INMT, while the AUC values of 14 compounds were negatively correlated with the expression of INMT. These findings suggest that INMT may affect prostate cancer’s occurrence, development, and drug sensitivity via various tumor signaling pathways and tumor microenvironments.

Gene therapy of prostate cancer using liposomes containing perforin expression vector driven by the promoter of prostate-specific antigen gene

Perforin secreted from cytotoxic lymphocytes plays a critical role in cancer immunosurveillance. The aim of this study was to investigate the therapeutic potential of liposomes containing perforin expression vector driven by the promotor of prostate-specific antigen (PSA). The anti-tumor effect of perforin was analyzed using prostate cancer (PC) PC-3 cells in which perforin expression was controlled by Tet-on system (PC-3PRF cells). Liposomes encapsulating PSA promoter-driven perforin expression vector (pLipo) were constructed for its specific expression in PC. The anti-tumor effect of pLipo was evaluated in vitro using docetaxel-resistant PC 22Rv1 PC cell line, 22Rv1DR, and PC-3 cells in the presence of human peripheral blood mono nuclear cells (PBMCs) and also in vivo using male nude mice bearing 22Rv1DR cell-derived tumor xenograft. Induction of perforin significantly inhibited growth of PC-3PRF cells. Treatment with pLipo induced perforin expression in 22Rv1DR cells expressing PSA but not in PC-3 cells lacking it. Treatment with pLipo at a low concentration was prone to inhibit growth of both cell lines and significantly inhibited growth of 22Rv1DR cells when co-incubated with PBMCs. The combined use of pLipo at a high concentration with PBMCs showed nearly complete inhibition of 22Rv1DR cell growth. Intravenous administration of pLipo via tail vein increased the level of perforin in tumor and serum and significantly decreased the tumor volume. Our results suggest that liposome-mediated PC-specific expression of perforin could be a novel therapy for advanced PC.

Antiandrogenic activity of Riboflavin 5′-phosphate (FMN) in 22Rv1 and LNCaP human prostate cancer cell lines

The androgen receptor is a hormone activated transcription factor that regulates the development and maintenance of male characteristics and represents one of the most well-established drug targets, being implicated not only in prostate cancer but also in many non-cancerous human diseases including androgenetic alopecia, acne vulgaris, and hirsutism. In this study, the antiandrogenic effects of FMN were investigated in 22Rv1 and LNCaP prostate cancer cells. FMN inhibited dihydrotestosterone (DHT)-induced protein expression of androgen receptor in 22Rv1cells. In another prostate cancer LNCaP cells, FMN decreased the protein level of DHT-induced prostate specific antigen (PSA). In addition, FMN downregulated DHT-induced mRNA expression of androgen regulated genes in both cell lines, showing less prominent inhibition in 22Rv1cells where androgen receptor had been significantly decreased by FMN. FMN was found to bind androgen receptor, demonstrating that it acted as a competitive androgen receptor antagonist. FMN increased the phosphorylation of Akt in 22Rv1 cells and this increment was abrogated by PI3K inhibitor wortmannin, resulting in a rescued androgen receptor protein level which was decreased by FMN. Additionally, FMN was found to increase the mRNA and protein level of E3 ligase mouse double minute 2. Our data suggest that the androgen receptor signaling is regulated through PI3K-Akt-MDM2 pathway in 22Rv1 cells. Together, our results indicate that FMN facilitated the degradation of androgen receptor in 22Rv1 cells and inhibited the expression of androgen regulated genes by competing the binding of DHT to androgen receptor in LNCaP cells, demonstrating its therapeutic potential as an antiandrogen.

EEFSEC knockdown inhibits proliferation, migration and invasion of prostate cancer cells in vitro

To investigate the role of selenocysteine-tRNA specific eukaryotic elongation factor (EEFSEC) in regulating the proliferation, migration, and invasion of human prostate cancer 22Rv1 cells. We detected EEFSEC mRNA expression levels in human normal prostate cell line RWPE1 and human prostate cancer cell lines 22Rv1, LNCaP, Vcap and PC-3 using qRT-PCR and EEFSEC protein expression in surgical specimens of prostate cancer and adjacent tissues using Western blotting. 22Rv1 cells were infected with a lentiviral vector carrying EEFSEC shRNA or a control lentivirus and the interference efficiency was determined using Western blotting. XTT assay was used to assess the changes in the viability of the infected cells, and Transwell chamber assay was used to examine the changes in cell migration and invasion. The effect of EEFSEC knockdown on cell cycle progression was determined with flow cytometry and by detecting the expressions of cell cycle proteins using qRT-PCR. EEFSEC was significantly upregulated in prostate cancer cells (P < 0.05), and a high expression of EEFSEC was associated with a poor prognosis of the patients with prostate cancer. In 22Rv1 cells, EEFSEC knockdown significantly suppressed the proliferation (P < 0.001), migration (P < 0.001) and invasion (P < 0.001) of the cells, resulted in cell cycle arrest in G0/G1 phase, obviously inhibited the expression of C-myc and CCNB1, and significantly increased the expression of p15. EEFSEC knockdown can inhibit the proliferation, migration, and invasion of prostate cancer cells in vitro possibly by down-regulating the expression of C-myc.

The Dual Effect of the HDAC Inhibitor Romidepsin on Androgen Receptor Signaling and DNA Damage Repair in Prostate Cancer.

Aberrant epigenetic modifications play an important role in prostate cancer development and progression. Histone deacetylase inhibitors (HDACi) are a novel class of small-molecular compounds that have been shown to have activity in several tumor types. Some HDACi's induce a stress response that affects DNA damage repair (radiosensitization) and androgen receptor (AR) signaling (increased sensitivity to androgen blockage). We hypothesized that the HDACi, romidepsin, would increase prostate cancer cell killing through dual effects on AR signaling and radiation DNA damage response.The prostate cancer cell lines LNCaP (androgen sensitive) and 22Rv1 (androgen insensitive) were treated with romidepsin (10 nM) plus or minus 2 Gy radiation. The added effect of enzalutamide (10 mM) was also tested. Cell viability was performed by Real-time Cell assay (RTCA), whole-transcriptome analysis by RNA sequencing, western blot analyses, immunofluorescent quantification of γ-H2AX double strand break foci, and colonogenic survival. Western blot analysis was used to measure the expression of (i) AR signaling pathway proteins that included wild-type AR and its AR-V7 splice variant and (ii) DNA repair genes that included Rad51, DNA-PK and γ-H2AX.Romidepsin reduced cell viability by RTCA of both cell lines in a dose dependent manner. The addition of enzalutamide further reduced cell viability and clonogenic survival. Gene ontology analysis from RNA-Seq data in both LNCaP and 22Rv1 cells revealed that romidepsin suppressed pathways related to prostate cancer aggressiveness including the AR, MYC, DNA damage response, and cell cycle progression pathways. By western blot analysis, romidepsin suppressed AR (LNCaP) and AR-V7 (22Rv1) expression and reduced DNA repair genes including RAD51, DNA-PK and phospho-ATM levels. After 2 Gy radiation, we found that treatment with 10nM romidepsin for 24 hours resulted in an increase in γ-H2AX foci in 35.2% in LNCaP and 27% in 22Rv1 cells respectively. By colonogenic assay, 10 nM romidepsin for 24 hours prior to 2 Gy to 8 Gy radiation resulted in a significant reduction in the surviving fractions. The addition of enzalutamide resulted in the greatest reduction in colonogenic survival.Our study demonstrated for the first time that HDACi romidepsin exerted dual effects on AR signaling and DNA damage response genes in androgen sensitive and insensitive prostate cancer cell lines, and that there is an added suppressive effect with the addition of enzalutamide to HDAC inhibition plus radiation. The use of HDACi's have promise clinically for the treatment of high-risk prostate cancer, for which androgen deprivation plus radiotherapy are the standard treatments.