Antiproliferative Response Research Articles

Bisphenol A induces cell cycle arrest in primary and prostate cancer cells through EGFR/ERK/p53 signaling pathway activation.

Bisphenol A (BPA) belongs to the class of chemicals known as endocrine disruptors and has been also involved in the pathogenesis and progression of endocrine related cancer such as breast and prostate cancers. Here, we have investigated the effect of BPA in human prostate cancer LNCaP cells and in human non-transformed epithelial prostate EPN cells. Our data showed that BPA induces the down regulation of cyclin D1 expression and the upregulation of the cell cycle inhibitors p21 and p27, leading to cell cycle arrest. Interestingly, we found that the BPA anti-proliferative response depends on a strong and rapid activation of epidermal growth factor receptor (EGFR), which stimulates ERK-dependent pathway. This, in turn, induces expression of p53 and its phosphorylation on residue Ser15, which is responsible for cell cycle arrest. EGFR activation occurs upon a cross talk with androgen (AR) and estradiol receptor-β (ERβ) which are known to bind BPA.Altogether, these findings show a novel signaling pathway in which EGFR activation plays a key role on BPA-induced cell cycle inhibition through a pathway involving AR and ERβ/EGFR complexes, ERK and p53. Our results provide new insights for understanding the molecular mechanisms in human prostate cancer. On the other, they could allow the development of new compounds that may be used to overcome human prostate cancer resistance to endocrine therapy in promising target therapeutic approaches.

1,25-Dihydroxyvitamin D3 inhibits the proliferation of rat mesangial cells induced by high glucose via DDIT4.

1,25-Dihydroxyvitamin D3(1,25(OH)2 D3) is a secosteroid with antiproliferative property. It also plays a pivotal renoprotective role in diabetic nephropathy. We investigated whether 1,25(OH)2D3 could inhibit the proliferation of rat mesangial cells exposed to high glucose via the DNA-damage-inducible transcript 4/mammalian target of rapamycin(DDIT4/mTOR) pathway. The cell proliferation rate and cell cycle duration were measured using cell counting kit-8 assay and flow cytometry. Protein expression was assayed by Western blot. Glucose acted as a growth factor in rat mesangial cells, promoted cell proliferation. In parallel, the protein expression of DDIT4, TSC1/TSC2, and 4E-BP1 were decreased, and Rheb, mTOR, and p70S6K were increased. Acting via the DDIT4/mTOR signaling, 1,25(OH)2 D3 treatment reversed these pathological changes, upregulated DDIT4, TSC1/TSC2, and 4E-BP1, downregulated Rheb, mTOR, and p70S6K. The short-term overexpression of DDIT4 inhibited the proliferation of rat mesangial cells, similar to 1,25(OH)2 D3 treatment. siRNA knockdown of DDIT4 suppressed antiproliferative responses to 1,25(OH)2 D3. These results suggest that 1,25(OH)2 D3 inhibits the proliferation of rat mesangial cells induced by high glucose via the DDIT4/mTOR signaling pathway.

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca2+ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.

Molecular docking of selected phytoconstituents with signaling molecules of Ultraviolet-B induced oxidative damage.

The signaling molecules TNF-α, AP-1, and NF-κB act to integrate multiple stress signals into a series of diverse antiproliferative responses. Disruption of these processes can promote tumor progression and chemoresistance. Naturally occurring plant derived compounds are considered as attractive candidates for cancer treatment and prevention. Phytoconstituents can control and modify various biological activities by interacting with molecules involved in concerned signaling pathways. The aim of this study was to find binding conformations between phytoconstituents and these signaling molecules responsible for multiple stress signals of UVB induced photodamage. Induced fit docking was carried out for understanding the binding interactions of pantothenic acid (vitamin B5); 3,4,5-trihydroxy benzoic acid (gallic acid); madecassic acid and hexadecanoic acid, ethyl ester (palmitic acid) with TNF-α, AP-1, and NF-κB. Favorable binding conformations between these signaling molecules and the four phytoconstituents were observed. A number of poses were generated to evaluate the binding conformations and common interacting residues between the ligands and proteins. Among them, the best ligands against TNF-α, AP-1, and NF-κB are reported. The present investigation strongly suggests the probable use of these flavonoids for the amelioration of UVB induced photodamage.

Melatonin enhances the apoptotic effects and modulates the changes in gene expression induced by docetaxel in MCF‑7 human breast cancer cells.

Results from clinical trials and multiple invivo and invitro studies point to melatonin as a promising adjuvant molecule with many beneficial effects when concomitantly administered with chemotherapy. Melatonin palliates side‑effects and enhances the efficacy of chemotherapeutic agents. However, the mechanisms through which melatonin regulates molecular changes induced by chemotherapeutic agents remain largely unknown. In this study, we demonstrated that melatonin enhanced the anti-proliferative and apoptotic responses to low doses of docetaxel in breast cancer cells. Importantly, these effects were more potent when melatonin was added prior to docetaxel. Treatment with 1µM docetaxel (equivalent to the therapeutic dosage) induced changes in gene expression profiles and melatonin modulated these changes. Specifically, docetaxel downregulated TP53, cyclin-dependent kinase inhibitor1A (CDKN1A) and cadherin 13 (CDH13), and upregulated mucin 1 (MUC1), GATA binding protein 3 (GATA3) and c-MYC, whereas melatonin counteracted these effects. Melatonin further stimulated the expression of the pro-apoptotic BAD and BAX genes, and enhanced the inhibition of the anti-apoptotic gene BCL-2 induced by docetaxel. The findings of this study suggest that melatonin is a molecule with potential for use as an adjuvant in cancer chemotherapy, which may have implications for designing clinical trials using chemotherapeutic drugs in combination with melatonin.

Capsaicin reduces genotoxicity, colonic cell proliferation and preneoplastic lesions induced by 1,2-dimethylhydrazine in rats

Capsaicin (8-Methyl-N-vanillyl-(trans)-6-nonenamide) is the major pungent ingredient found in chili peppers consumed worldwide. Most reports on capsaicin potential carcinogenicity have yielded inconsistent findings. Some studies have shown that capsaicin exerts anti-proliferative and pro-apoptotic effects on different cancer cell lines, while others have reported an association between capsaicin at high doses with mutagenicity and carcinogenicity. Thus, this study aimed at assessing the effects of capsaicin administration on 1,2-dimethyl-hydrazine (DMH)-induced colon carcinogenesis in male Wistar rats. Our results show that capsaicin administration, before and during carcinogen exposure, modified DMH-induced cytotoxicity and genotoxicity, promoting anti-proliferative and pro-apoptotic responses through the expression of the genes involved in apoptosis, cell cycle suppression and cell/tissue differentiation. Furthermore, capsaicin reduced aberrant crypt foci (ACF) number and multiplicity, although there were no differences in tumor incidence and multiplicity among the groups. Taken together, the results suggest that capsaicin may have a preventive effect against DMH-induced colorectal carcinogenesis.

Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors

Background/Aims: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo.Methods: By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. Results: The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. Conclusion: In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.

RNA interference-mediated knockdown of SIRT1 and/or SIRT2 in melanoma: Identification of downstream targets by large-scale proteomics analysis

Melanoma is the most notorious and fatal of all skin cancers and the existing treatment options have not been proven to effectively manage this neoplasm, especially the metastatic disease. Sirtuin (SIRT) proteins have been shown to be differentially expressed in melanoma. We have shown that SIRTs 1 and 2 were overexpressed in melanoma and inhibition of SIRT1 imparts anti-proliferative responses in human melanoma cells. To elucidate the impact of SIRT 1 and/or 2 in melanoma, we created stable knockdowns of SIRTs 1, 2, and their combination using shRNA mediated RNA interference in A375 human melanoma cells. We found that SIRT1 and SIRT1&2 combination knockdown caused a decreased cellular proliferation in melanoma cells. Further, the knockdown of SIRT 1 and/or 2 resulted in a decreased colony formation in melanoma cells. To explore the downstream targets of SIRTs 1 and/or 2, we employed a label-free quantitative nano-LC-MS/MS proteomics analysis using the stable lines. We found aberrant levels of proteins involved in many vital cellular processes, including cytoskeletal organization, ribosomal activity, oxidative stress response, and angiogenesis. These findings provide clear evidence of cellular systems undergoing alterations in response to sirtuin inhibition, and have unveiled several excellent candidates for future study. SignificanceMelanoma is the deadliest form of skin cancer, due to its aggressive nature, metastatic potential, and a lack of sufficient treatment options for advanced disease. Therefore, detailed investigations into the molecular mechanisms of melanoma growth and progression are needed. In the search for candidate genes to serve as therapeutic targets, the sirtuins show promise as they have been found to be upregulated in melanoma and they regulate a large number of proteins involved in cellular processes known to affect tumor growth, such as DNA damage repair, cell cycle arrest, and apoptosis. In this study, we used a large-scale label-free comparative proteomics system to identify novel protein targets that are affected following knockdown of SIRT1 and/or 2 in A375 metastatic melanoma cell line. Our study offers important insight into the potential downstream targets of SIRTs 1 and/or 2. This may unravel new potential areas of exploration in melanoma research.

A preclinical evaluation of the MEK inhibitor refametinib in HER2-positive breast cancer cell lines including those with acquired resistance to trastuzumab or lapatinib.

PurposeThe MEK/MAPK pathway is commonly activated in HER2-positive breast cancer, but little investigation of targeting this pathway has been undertaken. Here we present the results of an in vitro preclinical evaluation of refametinib, an allosteric MEK1/2 inhibitor, in HER2-positive breast cancer cell lines including models of acquired resistance to trastuzumab or lapatinib.MethodsA panel of HER2-positive breast cancer cells were profiled for mutational status and also for anti-proliferative response to refametinib alone and in combination with the PI3K inhibitor (PI3Ki) copanlisib and the HER2-targeted therapies trastuzumab and lapatinib. Reverse phase protein array (RPPA) was used to determine the effect of refametinib alone and in combination with PI3Ki and HER2-inhibitors on expression and phosphorylation of proteins in the PI3K/AKT and MEK/MAPK pathways. We validated our proteomic in vitro findings by utilising RPPA analysis of patients who received either trastuzumab, lapatinib or the combination of both drugs in the NCT00524303/LPT109096 clinical trial.ResultsRefametinib has anti-proliferative effects when used alone in 2/3 parental HER2-positive breast cancer cell lines (HCC1954, BT474), along with 3 models of these 2 cell lines with acquired trastuzumab or lapatinib resistance (6 cell lines tested). Refametinib treatment led to complete inhibition of MAPK signalling. In HCC1954, the most refametinib-sensitive cell line (IC50 = 397 nM), lapatinib treatment inhibits phosphorylation of MEK and MAPK but activates AKT phosphorylation, in contrast to the other 2 parental cell lines tested (BT474-P, SKBR3-P), suggesting that HER2 may directly activate MEK/MAPK and not PI3K/AKT in HCC1954 cells but not in the other 2 cell lines, perhaps explaining the refametinib-sensitivity of this cell line. Using RPPA data from patients who received either trastuzumab, lapatinib or the combination of both drugs together with chemotherapy in the NCT00524303 clinical trial, we found that 18% (n=38) of tumours had decreased MAPK and increased AKT phosphorylation 14 days after treatment with HER2-targeted therapies. The combination of MEK inhibition (MEKi) with refametinib and copanlisib led to synergistic inhibition of growth in 4/6 cell lines tested (CI @ED75 = 0.39-0.75), whilst the combinations of lapatinib and refametinib led to synergistic inhibition of growth in 3/6 cell lines (CI @ED75 = 0.39-0.80).ConclusionRefametinib alone or in combination with copanlisib or lapatinib could represent an improved treatment strategy for some patients with HER2-positive breast cancer, and should be considered for clinical trial evaluation. The direct down-regulation of MEK/MAPK but not AKT signalling by HER2 inhibition (e.g. by lapatinib or trastuzumab), which we demonstrate occurs in 18% of HER2-positive breast cancers may serve as a potential biomarker of responsiveness to the MEK inhibitor refametinib.

Abstract 1947: Combination of Vincristine and Clotam induces antiproliferative response in medulloblastoma cells

Abstract Medulloblastoma (MB) is the most common pediatric malignant brain tumor and usually originates in the cerebellum. These tumors have the propensity to disseminate throughout the central nervous system and are often difficult to treat. Chemotherapy is widely accepted as part of the multimodality treatment approach for MB. However, it is associated with debilitating toxicity and potential long term disabilities. Vincristine is a commonly used chemotherapeutic agent for MB treatment. This drug is known to induce some toxic effects including peripheral neuropathy. The aim of this study was to test a combination treatment involving Vincristine and an anti-cancer non-steroidal anti-inflammatory drug, Clotam (Tolfenamic acid) against MB cell lines. Previously, we showed that Clotam inhibited MB cell proliferation and tumor growth in mice by targeting the transcription factor, Specificity protein1 (Sp1) and an inhibitor of apoptosis protein, BIRC5 (baculoviral inhibitor of apoptosis repeat-containing 5). The overexpression of BIRC5 is associated with aggressiveness and poor prognosis in several cancers. MB cells, DAOY and D283 cells were treated with vehicle (dimethyl sulfoxide) or low dose of Vincristine (DAOY: 2ng/ml; D283: 1ng/ml) or Clotam (DAOY & D283: 10 µg/ml) or combination of Vincristine + Clotam and the cell viability was measured at 1 and 2 days post-treatment using Cell-TiterGlo kit. Flow cytometry was employed to analyze apoptotic cells using Annexin-V staining and cell cycle phase distribution using propidium iodide staining. The activation of apoptotic pathways was further investigated by assessing the levels of effector caspases with CaspaseGlo kit and the expression of apoptotic markers [cleaved Poly (ADP-ribose) polymerase (c-PARP), B-cell lymphoma 2, and BIRC5] by Western blot analysis. The expression of key proteins associated with cell cycle (Cyclin A, B, D, CDK4/6, and p21) was also determined by Western blot analysis. When compared to individual agents, the combination of Clotam and Vincristine increased MB cell growth inhibition which is accompanied by an induction of apoptotic markers and the modulation of (Cyclin A, B, D and CDK4/6: down-regulation; p21: up-regulation) proteins associated with cell cycle phase distribution. These results suggest that Vincristine and Clotam combination treatment is effective for inducing anti-proliferative effect in MB cells. The experiments to evaluate the effect of this combination in animal model for MB are currently under study. Citation Format: Shruti V. Patil, Don Eslin, Robert Sutphin, Umesh T. Sankpal, Yazmin Hernandez, Areeba Hafeez, W. Paul Bowman, Riyaz Basha. Combination of Vincristine and Clotam induces antiproliferative response in medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1947. doi:10.1158/1538-7445.AM2017-1947

Abstract 5878: Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway

Abstract Trichostatin A (TSA), a potent histone deacetylase (HDAC) inhibitor, has been reported to elicit anti-proliferative response in various tumors. Here, we investigated antitumor effect of TSA alone or in combination with conventional chemotherapeutic agents on urothelial carcinoma (UC) cells. We use one high-grade UC cell line (T24) and another UC cells (NTUUC) obtained by primary culture from the surgical specimen of a women with high-grade and metastatic bladder UC. The cytotoxicity and apoptosis induced by TSA alone , chemotherapeutic agents (cisplatin, gemcitabine and doxorubicin) and combined treatment were assessed by MTT assay and fluorescence-activated cell sorting and flow cytometry. The expression of phosphor c-Raf, phosphor-MEK1/2, and phosphor-ERK1/2 were measured by Western blot. urther elucidation on the role of Raf/MEK/ERK pathoway on the TSA-enhanced cytoxicity were exmained by using ERK1 siRNA knockdown and the specific MEK inhibitor (PD98059). Our results showed TSA markedly enhances the cytotoxicity and apoptosis of three chemotherapeutic agents in UC cells with concurrent suppression of Raf/MEK/ERK signaling pathway. Consistently, inhibition of Down-regulation of ERK by MEK inhibitor or by ERK 1 siRNA knockdown potentiated the chemotherapeutic agnet-induced cytotoxicity of TSA in UC cells. We concluded that TSA potentiates the therapeutic efficacy of cisplatin, gemcitabine and doxorubicin in human UC cells through Raf/MEK/ERK signaling pathway. These findings provide a new treatment strategy against UC. Note: This abstract was not presented at the meeting. Citation Format: Kuan-Lin Kuo, Chung-Sheng shi, Ju-Tong Hsieh, Shiang-Peng Chen, Wei-Chou Lin, Shih-Ming Liao, Shing-Hwa Liu, Kuo-How Huang. Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5878. doi:10.1158/1538-7445.AM2017-5878

Abstract LB-151: The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizer inducing tumor cell death through the spindle assembly checkpoint. BAL101553 has anti-cancer activity in diverse tumor models refractory to standard therapies, and is currently undergoing Phase 1/2a evaluation in advanced cancer patients with IV (weekly) and oral (daily) dosing schedules. Here, the vascular disruption activity of BAL101553 was evaluated in the context of administration route and implications for combinations with VEGF-targeted therapies. Methods: MTD IV (day[d] 1) and oral (d1 & d1 - 4) dosing regimens were tested in H460 NSCLC xenografts. Tumors were analyzed by IHC for proliferation (Ki67) and vascularization (CD31, lectin perfusion). Drug combinations were performed in Pgp-overexpressing, colon carcinoma SW480 xenografts using BAL101553 IV 1xweek (21.3 mg/kg) or oral daily (15 mg/kg) and bevacizumab IP d1 & 4 (5 mg/kg). Results: H460 tumor-bearing mice received once weekly (IV 25 mg/kg; oral 60 mg/kg; high Cmax regimens) or daily (oral 25 mg/kg; low Cmax, high AUC) BAL101553 regimens which elicited equipotent anti-tumor effects (ΔT/Cs = 26-34%). Although all treatments elicited a similar anti-proliferative response (% reduction of mean Ki67 signal+/-SD=10+/-1.2; p<0.05 vs. controls), tumor vessel density was most profoundly reduced by high Cmax regimens. Interestingly, daily-oral treatment (low Cmax, high AUC) had reduced tumor anti-vascular activity, while presenting equivalent anti-proliferative and anti-tumor efficacy. In vitro assays against tumor cell lines showed that sub-cytotoxic BAL27862 concentrations reduced HIF-1α protein levels in normoxic and hypoxic conditions, diminishing VEGF-expression and secretion, indicating direct interference of the drug on hypoxic adaptation and endothelial tube formation. Based on these data, combination of the anti-VEGF antibody bevacizumab with BAL101553 was assessed in the paclitaxel refractory SW480 tumor model, using MTD BAL101553 dosing (IV weekly & oral daily). All monotherapies induced an anti-tumor response (ΔT/Cs bevacizumab: 35%; IV BAL101553 22%; oral BAL101553: 53%). However, bevacizumab combined with IV or oral BAL101553 was superior to single agents (ΔT/C: 6% and 17%, resp., p<0.05 vs. control) with IV combination associated with the least functional tumor vasculature. Conclusions: BAL101553 targets tumor cell proliferation, tumor hypoxic adaptation and vascularization. The latter is Cmax-driven and can be attenuated by daily oral dosing, providing an alternative pharmacodynamic endpoint with implications for clinical treatment strategies. IV and orally administered BAL101553 both elicit superior anti-tumor responses in combination with anti-VEGF therapy. Citation Format: Ashish Sharma, Felix Bachmann, Angela Broggini-Tenzer, Matthias Guckenberger, Heidi Lane, Martin N. Pruschy. The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2017-LB-151

Abstract 5415: Targeted depletion of polo-like kinase 1 alters metabolic regulation in melanoma

Abstract Polo-like kinase 1 (PLK1), a critical regulator of mitosis, is shown to be overexpressed in a variety of cancers. PLK1 overexpression has also been linked to poor disease prognosis and a lessened survival in cancer patients. Previously, we have shown that PLK1 is overexpressed in human melanoma and its inhibition causes significant anti-proliferative response in vitro (in multiple melanoma cells) as well as in vivo (in human melanoma xenografts). Further, in another study we demonstrated, based on a large-scale label-free comparative proteomics analysis, that PLK1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in human melanoma cells resulted in an alteration of certain metabolism-associated proteins with an associated decrease in cellular metabolism. In this study, to further explore the association between PLK1 and cellular metabolism, we utilized a doxycycline-inducible PLK1 knockdown approach in A375 melanoma cells coupled with a Human Glucose Metabolism PCR array that covers 84 key genes involved in the regulation and enzymatic pathways of glucose and glycogen metabolism. We found that PLK1 knockdown resulted in a significant downregulation of 29 genes and upregulation of 3 genes (more than 2 fold change) associated with cellular metabolism. IDH1, PDP2 and PCK1 were >3-fold downregulated while FBP1 was >7-fold upregulated. Through Ingenuity Pathway Analysis (IPA), we identified glycolysis and the pentose phosphate pathway as major canonical pathways altered by PLK1 inhibition. Further, IPA identified that PLK1 inhibition-modulated genes were largely associated with the proliferation of cells, where FBP1 appeared as a key regulatory player. PCK1 was found to be highly downregulated in our array, and IPA identified it as part of a monosaccharide regulation network. We further validated our data in vivo and found that BI 6727 treatment resulted in a decrease in PCK1 and increase in FBP1 in A375 melanoma cell implanted xenografts. In addition, we observed a strong inverse correlation between PLK1 and FBP1 in multiple melanoma cell lines, with FBP1 expression significantly downregulated in a panel of melanoma cells compared to normal melanocytes. Moreover, BI 6727 treatment resulted in an upregulation in FBP1 in A375, Hs 294T and G361 melanoma cells. Interestingly, in recent studies, FBP1 (fructose-bisphosphatase) that is a rate-limiting enzyme in gluconeogenesis, has been shown to function as a tumor suppressor in certain cancers. Overall, our study support the hypothesis that PLK1 is a regulator of metabolism maintenance that affects the melanoma cell growth. Citation Format: Rosie E. Gutteridge, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. Targeted depletion of polo-like kinase 1 alters metabolic regulation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5415. doi:10.1158/1538-7445.AM2017-5415

Abstract 3195: DYRK1B inhibitors prevent pharmacologic quiescence and sensitize lung cancers to EGFR inhibitors

Abstract Many oncoproteins that activate signaling dysregulate cell growth by activating the cyclinD/cdk4, cdk6 complex and abrogate in the G1 checkpoint (mutant RAS, BRAF, PIK3CA, EGFR etc.) Inhibition of these oncoproteins inhibits cyclinD expression and kinase activity as well as RB phosphorylation and results in G0/G1 arrest and apoptosis. It has been posted that cells arrested in G0/G1 are protected from cell death. DYRK1B kinase plays an important role in physiologic quiescence by maintaining the viability of cells arrested in the G0/G1 phases of the cell cycle. DYRK1B regulates the G0/G1 transition by phosphorylating cyclin D1 and p27, whereby destabilizing cyclin D1 and stabilizing p27. The balance between these two proteins is essential for maintenance of cells in G0 (quiescent) state. DYRK1B is sparsely expressed in healthy tissues yet is overexpressed in patient samples with variety of neoplasms, including NSCLC. Cancer and normal cells exhibit differential sensitivity to DYRK1B inhibition. Whereas DYRK1B appears essential for survival of cancer cells either endogenously expressed or upregulated, its inhibition in healthy cells was not cytotoxic and had no effect on the cell cycle distribution. We hypothesized that if DYRK1B is required for maintenance of quiescence, its inhibition would enhance the effects of inhibitors of oncogenic signaling. We used a selective, ATP competitive DYRK1B inhibitor to test this idea. EGFR inhibitors are effective in non-small cell lung cancers with mutant EGFR and induce G0/G1 block and some apoptosis. These drugs significantly extend survival, but acquired resistance almost always supervenes. In vitro, the anti-proliferative response of lung tumors with mutant EGFR to EGFR inhibitors was shown to be primarily due to the entry of cells into a quiescent (G0) state associated with an induction of DYRK1B expression. Moreover, addition of a DYRK1B inhibitor reversed the quiescent state in cells exposed to EGFR inhibitors. Combined therapy of EGFR TKIs and the DYRK1B inhibitor significantly enhanced the antitumor response compare to either drug alone. Specifically, it is shown that treatment with osimertinib results in an increase of cells in G0 state: 63% as compared to non-treated cells, 29%. The induction of quiescence is prevented when cells are treated with the combination of osimertinib and DYRK1B inhibitor and massive apoptosis is observed: 62% compared to 12% with osimertinib alone. It is important to note that co-treatment with DYRK1B inhibitor not only prevented the entry of cancer cells into quiescence but also depleted pre-existent reservoir of quiescent cancer cells. The results suggest that inhibition of EGFR causes tumor cells to enter into a DYRK1B-dependent quiescent state in which DYRK1B is required for their survival. Inhibition of DYRK1B dramatically enhances the antitumor activity of EGFR inhibition and may improve outcome in patients. Citation Format: Maria Vilenchik, Alexandra Kuznetsova, Michael Frid, Yuriy Gankin, Marc Duey, Neal Rosen. DYRK1B inhibitors prevent pharmacologic quiescence and sensitize lung cancers to EGFR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3195. doi:10.1158/1538-7445.AM2017-3195

Abstract 820: Potential pro-proliferative role of SIRT6 in melanoma

Abstract Melanoma is one of the deadliest form of skin cancer that can rapidly metastasize to become lethal, if not diagnosed early or left untreated. In 2016, approximately 76,380 new melanoma cases and 10,130 melanoma-related deaths are predicted in the United States. Current preventive and therapeutic strategies have not been sufficiently effective in the management of melanoma. Therefore, novel molecular targets and treatments are required for an effective management of this neoplasm. In our laboratory, we are assessing the role and functional and therapeutic significance of sirtuin family of proteins in melanoma. Sirtuins (SIRTs) have been conserved through evolution from prokaryotic to eukaryotic cells. SIRTs are nicotinamide adenine dinucleotide (NAD+) dependent protein deacetylases and belong to class III of the histone deacetylase (HDAC) family. Seven members of the mammalian SIRT family are known to date, and despite structural similarities, each SIRT has their own biological niche, performing unique functions via regulating critical mechanisms in the cell. The role of SIRTs in cancer is somewhat controversial, as they have exhibited conflicting functions (tumor promoter vs. tumor suppressor) depending on cell and tissue contexts. The sirtuin SIRT6, a predominantly nuclear protein, has been shown to conduct ADP-ribosyl transferase and histone deacetylase activities. SIRT6 plays key roles in DNA repair, inflammation and metabolic diseases such as cancer. Currently, the role of SIRT6 in melanoma is not known. The objective of this study was to determine the role of SIRT6 in melanoma. Using a panel of human melanoma cell lines (A375, Hs 294T, G361, SK-MEL-2, SK-MEL-28, SK-MEL-31, WM115 and WM35) differing in genetic complexity and disease progression stage, and normal human epidermal melanocytes (NHEMs), we determined the endogenous expression levels of SIRT6. We found that compared to NHEMs, SIRT6 is significantly upregulated in melanoma cell lines, at mRNA as well as protein levels, as shown by quantitative Real-Time PCR and western blot analyses. Further, employing a human tissue microarray (TMA) coupled with quantitative Vectra™ analysis, we determined the expression profile of SIRT6 protein in human melanoma and melanocytic nevus tissues. Our data demonstrated that SIRT6 is significantly overexpressed in human melanoma tissues when compared to nevi. Furthermore, lentiviral short hairpin RNA-mediated knockdown of SIRT6 in human melanoma cells was found to result in a marked anti-proliferative response in melanoma cells. Taken together, our data suggest that SIRT6 overexpression could potentially be a contributing factor in melanoma progression. Further detailed studies are underway to understand the functional significance of SIRT6 in melanoma development and progression. Citation Format: Liz Garcia-Peterson, Mary A. Ndiaye, Chandra K. Singh, Wei Huang, Nihal Ahmad. Potential pro-proliferative role of SIRT6 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 820. doi:10.1158/1538-7445.AM2017-820

Unphosphorylated STAT3 regulates the antiproliferative, antiviral, and gene-inducing actions of type I interferons

Type I interferon (IFNα/β) induces antiviral and antiproliferative responses in cells through the induction of IFN-stimulated genes (ISGs). Although the roles of IFN-activated STAT1 and STAT2 in the IFN response are well described, the function of STAT3 is poorly characterized. We investigated the role of STAT3 in the biological response to IFNα/β in mouse embryonic fibroblasts (MEFs) with a germ line deletion of STAT3. These STAT3 knockout (STAT3-KO) MEFs were reconstituted with STAT3 or the F705-STAT3 mutant (unphosphorylated STAT3) where the canonical Y705 tyrosine phosphorylation site was mutated. We show that both STAT3 and unphosphorylated STAT3 expression enhance the sensitivity of MEFs to the antiviral, antiproliferative and gene-inducing actions of IFN. By chromatin immunoprecipitation assays, unphosphorylated STAT3 appears to bind, albeit weakly, to select gene promoters to enhance their expression. These results suggest that unphosphorylated STAT3 plays an important role in the IFN response pathway.

The inhibition of FGF receptor 1 activity mediates sorafenib antiproliferative effects in human malignant pleural mesothelioma tumor-initiating cells

BackgroundMalignant pleural mesothelioma is an aggressive cancer, characterized by rapid progression and high mortality. Persistence of tumor-initiating cells (TICs, or cancer stem cells) after cytotoxic drug treatment is responsible for tumor relapse, and represents one of the main reasons for the poor prognosis of mesothelioma. In fact, identification of the molecules affecting TIC viability is still a significant challenge.MethodsTIC-enriched cultures were obtained from 10 human malignant pleural mesotheliomas and cultured in vitro. Three fully characterized tumorigenic cultures, named MM1, MM3, and MM4, were selected and used to assess antiproliferative effects of the multi-kinase inhibitor sorafenib. Cell viability was investigated by MTT assay, and cell cycle analysis as well as induction of apoptosis were determined by flow cytometry. Western blotting was performed to reveal the modulation of protein expression and the phosphorylation status of pathways associated with sorafenib treatment.ResultsWe analyzed the molecular mechanisms of the antiproliferative effects of sorafenib in mesothelioma TIC cultures. Sorafenib inhibited cell cycle progression in all cultures, but only in MM3 and MM4 cells was this effect associated with Mcl-1-dependent apoptosis. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt, and STAT3 phosphorylation. These effects were abolished by sorafenib only in bFGF-treated cells, while a modest inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGF receptor (FGFR) inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib.Moreover, in MM1 cells, which release high levels of bFGF and showed autocrine activation of FGFR1 and constitutive phosphorylation/activation of MEK-ERK1/2, sorafenib induced a more effective antiproliferative response, confirming that the main target of the drug is the inhibition of FGFR1 activity.ConclusionsThese results suggest that, in malignant pleural mesothelioma TICs, bFGF signaling is the main target of the antiproliferative response of sorafenib, acting directly on the FGFR1 activation. Patients with constitutive FGFR1 activation via an autocrine loop may be more sensitive to sorafenib treatment and the analysis of this possibility warrants further clinical investigation.

Impact of 27-hydroxylase (CYP27A1) and 27-hydroxycholesterol in breast cancer.

The impact of systemic 27-hydroxycholesterol (27HC) and intratumoral CYP27A1 expression on pathobiology and clinical response to statins in breast cancer needs clarification. 27HC is an oxysterol produced from cholesterol by the monooxygenase CYP27A1, which regulates intracellular cholesterol homeostasis. 27HC also acts as an endogenous selective estrogen receptor (ER) modulator capable of increasing breast cancer growth and metastasis. 27HC levels can be modulated by statins or direct inhibition of CYP27A1, thereby attenuating its pro-tumorigenic activities. Herein, the effect of statins on serum 27HC and tumor-specific CYP27A1 expression was evaluated in 42 breast cancer patients treated with atorvastatin within a phase II clinical trial. Further, the associations between CYP27A1 expression with other primary tumor pathological features and clinical outcomes were studied in two additional independent cohorts. Statin treatment effectively decreased serum 27HC and deregulated CYP27A1 expression in tumors. However, these changes were not associated with anti-proliferative responses to statin treatment. CYP27A1 was heterogeneously expressed among primary tumors, with high expression significantly associated with high tumor grade, ER negativity and basal-like subtype. High CYP27A1 expression was independently prognostic for longer recurrence-free and overall survival. Importantly, the beneficial effect of high CYP27A1 in ER-positive breast cancer seemed limited to women aged ≤50 years. These results establish a link between CYP27A1 and breast cancer pathobiology and prognosis and propose that the efficacy of statins in reducing serum lipids does not directly translate to anti-proliferative effects in tumors. Changes in other undetermined serum or tumor factors suggestively mediate the anti-proliferative effects of statins in breast cancer.

Erratum to: ESR1 mutations affect anti-proliferative responses to tamoxifen through enhanced cross-talk with IGF signaling.

The purpose of this study was to address the role of ESR1 hormone-binding mutations in breast cancer. Soft agar anchorage-independent growth assay, Western blot, ERE reporter transactivation assay, proximity ligation assay (PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR (ddPCR), Kaplan–Meier analysis, and statistical analysis. It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers; however, we do not yet know how to best treat these patients. We have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and Western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R, and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR, we identified mutations at high frequencies ranging from 12 % for Y537N, 5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam.

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

BackgroundLung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment.MethodsThe human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity.ResultsWe demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment.ConclusionsOur data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.