Triple negative breast cancer (TNBC) is an aggressive disease with a 5-y relative survival rate of 11% after distant metastasis. To survive the metastatic cascade, tumor cells remodel their signaling ...pathways by regulating energy production and upregulating survival pathways. AMP-activated protein kinase (AMPK) and Akt regulate energy homeostasis and survival, however, the individual or synergistic role of AMPK and Akt isoforms during lung colonization by TNBC cells is unknown. The purpose of this study was to establish whether targeting Akt, AMPKα or both Akt and AMPKα isoforms in circulating cancer cells can suppress TNBC lung colonization. Transient silencing of Akt1 or Akt2 dramatically decreased metastatic colonization of lungs by inducing apoptosis or inhibiting invasion, respectively. Importantly, transient pharmacologic inhibition of Akt activity with MK-2206 or AZD5363 inhibitors did not prevent colonization of lung tissue by TNBC cells. Knockdown of AMPKα1, AMPKα2, or AMPKα1/2 also had no effect on metastatic colonization of lungs. Taken together, these findings demonstrate that transient decrease in AMPK isoforms expression alone or in combination with Akt1 in circulating tumor cells does not synergistically reduce TNBC metastatic lung colonization. Our results also provide evidence that Akt1 and Akt2 expression serve as a bottleneck that can challenge colonization of lungs by TNBC cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hepatocellular carcinoma (HCC) is the most common liver cancer and presents various degrees of aggressiveness. In this case study, we reported the management of an aggressive HCC patient who was a ...young immigrant from a hepatitis B endemic country with locally advanced HCC with portal involvement at presentation. Patient was initially managed with Yttrium-90 (Y-90) instillation, then systemic treatment when he had disease progression. Despite multiple lines of systemic treatments, patient continued to progress and developed significant cardiac involvement and pulmonary tumor thromboemboli. His course of treatment was further complicated by hemoptysis, presumably from hemorrhagic tumor thromboemboli. Patient became ineligible for systemic treatment due to the risk of hemoptysis, thus, subsequently managed with a course of palliative radiotherapy. Unfortunately, patient developed hemorrhagic shock, cardiac failure, and septic shock during radiation treatment and expired shortly afterward. In this case report, we discussed multi-modal treatments, including Y-90, systemic treatment, and radiotherapy, in managing complicated and aggressive HCC. We also reported risk factors, prognostic factors, efficacy of Y-90 instillation and the necessity of a personalized treatment approach. In conclusion, there is no consensus on managing patients with metastatic HCC with cardiac and pulmonary involvement currently. Treatment modalities are often highly personalized and require multi-disciplinary discussion.
Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is characterized by poor survival. Radiotherapy plays an important role in treating TNBC. The purpose of this ...study was to determine whether inhibiting the AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) pathways alone or in combination potentiates radiotherapy in TNBC. AMPKα1 and AMPKα2 knockdown diminished cyclin D1 expression and induced G1 cell cycle arrest but did not induce apoptosis alone or in combination with radiotherapy. Next, we analyzed the role of PI3K p85α, p85β, p110α, p110β, Akt1, and Akt2 proteins on TNBC cell cycle progression and apoptosis induction. Akt1 and p110α knockdown diminished cyclin D1 expression and induced apoptosis. Silencing Akt1 promoted synergistic apoptosis induction during radiotherapy and further reduced survival after radiation. Treatment with the Akt inhibitor, MK-2206 48 h after radiotherapy decreased Akt1 levels and potentiated radiation-induced apoptosis. Together, our results demonstrate that AMPKα, p110α, and Akt1 promote TNBC proliferation and that Akt1 is a key regulator of radiosensitivity in TNBC. Importantly, combining radiotherapy with the pharmacological inhibition of Akt1 expression is a potentially promising approach for the treatment of TNBC.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Patients with advanced-stage gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have a poor overall prognosis despite chemotherapy and radiotherapy (e.g., peptide receptor radionuclide therapy ...(PRRT)). Better treatment options are needed to improve disease regression and patient survival. The purpose of this study was to examine a new treatment strategy by combining PI3K/mTOR dual inhibition and radiotherapy. First, we assessed the efficacy of two PI3K/mTOR dual inhibitors, PF-04691502 and PKI-402, to inhibit pAkt and increase apoptosis in NET cell lines (BON and QGP-1) and patient-derived tumor spheroids as single agents or combined with radiotherapy (XRT). Treatment with PF-04691502 decreased pAkt (Ser473) expression for up to 72 h compared with the control; in contrast, decreased pAkt expression was noted for less than 24 h with PKI-402. Simultaneous treatment with PF-04691502 and XRT did not induce apoptosis in NET cells; however, the addition of PF-04691502 48 h after XRT significantly increased apoptosis compared to PF-04691502 or XRT treatment alone. Our results demonstrate that schedule-dependent administration of a PI3K/mTOR inhibitor, combined with XRT, can enhance cytotoxicity by promoting the radiosensitivity of NET cells. Moreover, our findings suggest that radiotherapy, in combination with timed PI3K/mTOR inhibition, may be a promising therapeutic regimen for patients with GEP-NET.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Introduction: Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from multiple organs. The mammalian Target of Rapamycin (mTOR) and phosphatidylinositol 3-kinase ...(PI3K)/Akt signaling pathway has a well-established role in NETs that was supported by the clinical efficacy, shown by the rapamycin analog, Everolimus. However, treatment with everolimus results in activation of Akt as an escape mechanism for cancer cells. Novel dual mTOR/PI3K inhibitors, PF-04691502 and PKI-402, were shown to have superior antineoplastic effect, however, not have been tested in NETs. The purpose of our current study was to test the effects of PF-04691502 and PKI-402 on proliferation, apoptosis and Akt activation of NET cells in vitro.
Methods: For these studies, we utilized two human NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) BON and QGP-1 cells were treated with increasing concentrations of PF-04691502 (100nM – 10,000nM) and PKI-402 (50nM – 1,000nM) at 72, 96 and 120 h to determine half maximal inhibitory concentration (IC-50) by sulforhodamine B (SRB) assay. (ii) BON and QGP-1 cells were then treated with and harvested 24 h later to further examine expression of cyclin D1 and PARP by Western blot. In addition, phospho-Akt (S473), phospho-S6 (S235/236), and phospho-4E-BP1(Thr37/46) expression was evaluated by Western blot to assess the effect of PI3K/mTOR inhibition on downstream signaling proteins in NET cells.
Results: (i) SRB assays demonstrated that IC-50 was 74nM for PF-04691502 in BON cells, 196nM for PF-04691502 in QGP-1 cells, 40nM for PKI-402 in BON cells, and 45nM for PKI-402 in QGP-1 cells at 96 h. (ii) Cyclin D1 expression was decreased in a dose-dependent fashion in both BON and QGP-1 cells. PARP expression was increased in QGP-1 cells treated with PF-04691502 (500nM). In contrast, we did not observe a significant increase in PARP expression in BON cells following PF-04691502 treatment or with PKI-402 treatment of either cell line. (iii) A dose-dependent decrease in p-AKT (S473) and p-6S (S235/236) expression was noted in cells treated with PF-04691502 at 24 h and with PKI-402 at 4 h. We did not observe significant difference in p4E-BP1(Thr37/46) expression in QGP-1 cells after treatment with PF-04691502 at 24h and PKI-402 at 4h.
Conclusion: Our results demonstrate that both PF-04691502 and PKI-402 have a cytostatic inhibitory effect on proliferation in both BON and QGP-1 cells. In addition, treatment with PF-04691502 causes cytotoxic induction of apoptosis in QGP-1 cells, but not in BON cells. Our results suggest that PI3K/mTOR inhibition with PF-04691502 could be a novel therapeutic approach to the treatment of NETs.
Citation Format: Zeta Chow, Courtney Townsend, B. Mark Evers, Piotr Rychahou. Effect of novel mTOR-PI3K dual inhibitors on neuroendocrine tumor cell proliferation and apoptosis abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 676.
Abstract
Background: Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from multiple organs. Peptide Receptor Radionuclide Therapy (PRRT) is a novel therapy approved for ...treating NETs with a treatment response rate of 20-30%. To improve therapeutic response of PRRT, 3-AP (Triapine), which is a ribonucleotide reductase (RR) inhibitor with antineoplastic and radiation sensitizing properties, can be used in combination with PRRT. The purpose of our study is to examine effects of Triapine treatment on cellular proliferation, apoptosis and activation of DNA repair pathway in NET cells in vitro.
Methods: For these studies, we utilized two human NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) To determine effective dose range of Triapine (500 - 2500nM), proliferation was measured at 48, 72, and 96 h with sulforhodamine B (SRB) assay. (ii) To evaluate the effects of radiosensitivity, cultures were treated with increasing doses (250 - 5,000nM), received 2 Gy radiation (XRT) 1h later, exposed to Triapine for 24 hours, rinsed, and fed Triapine-free media; colonies were counted and quantified with SRB assays 14 days later and survival curves were generated. (iii) To further characterize cellular responses, cells were treated with Triapine (250 - 5,000 nM), irradiated 1h later, and harvested 24 h later to examine expression levels of Cyclin D1, cleaved PARP, p-DNA-PK (S2056), p-ATM (S1981) and p-ATR (S428) by Western blot.
Results: (i) SRB proliferation assay demonstrated the effective dose range, determined by half maximal inhibitory concentration (IC-50), was 1,3 µM in BON cells and 1.73 µM in QGP-1 cells at 96 h. (ii) Exposure to Triapine before irradiation resulted in a significant increase in the radiosensitivity of each of the tumor cell lines with dose enhancement factors at a surviving fraction of 0.10 of 1.8 and 1.6 for BON and QGP-1, respectively. (iii) Triapine treatment alone activated DNA repair pathway in a dose-dependent fashion as determined by pDNA-PK (S2056) expression. Increasing p-ATM (S1981) expression was observed in a dose-dependent fashion in QGP-1 cells, but not in BON cells. In addition, p-ATR (S428) expression was not significantly different in either cell line. (iv) Triapine treatment alone or in combination with 2 and 4 Gy XRT resulted in dose-dependent decrease in cyclin D1 expression in both cell lines, however, no significant increase in cleaved PARP expression at 24 and 48h.
Conclusion: Our results demonstrated rapid and dose dependent decrease in cyclin D1 expression after Triapine treatment, dose-dependent activation of DNA-PK, the key component of the non-homologous end joining pathway and significant increase in the radiosensitivity of each of the tumor cell lines. In conclusion, our findings provide a rationale for inclusion of Triapine as a radiosensitizer in combination with PRRT in treating NETs.
Citation Format: Zeta Chow, Aman Chauhan, Lowell Anthony, Courtney Townsend, B. Mark Evers, Piotr Rychahou. Effect of 3AP on neuroendocrine tumor cell proliferation, apoptosis and activation of DNA repair pathway abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6269.
Abstract
Background: Peptide receptor radionuclide therapy (PRRT) is a novel radiotherapy approved for gastroenteropancreatic neuroendocrine tumors (GEP-NETs); however, PRRT has a relatively low ...treatment response rate of 20-30%. We hypothesize that addition of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a ribonucleotide reductase (RnR) inhibitor that inhibits the rate-limiting enzyme of DNA synthesis and repair, can improve radiotherapy outcomes. The purpose of this study is to determine the therapeutic rationale of RnR-targeted therapy and evaluate 3-AP as a radiosensitizer in treating GEP-NET in vitro and in vivo. Methods: We utilized two human GEP-NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) Cells were treated with 3-AP for 96h and cell proliferation assays were performed using a sulforhodamine B (SRB) kit. Next, cells were treated with 3-AP for 24, 48, 72, 96h and cell cycle analyses were performed with Beckman Coulter cell cycle kit and quantified by flow cytometry. Expression of cell cycle arrest markers (i.e., pCdk-1, cyclin D1) was assessed by immunoblotting. (ii) To determine radiosensitivity in vitro, cells were treated with 3-AP for 16h, followed by X-ray radiotherapy (XRT), and incubation in drug-free medium. Cell survival was assessed by colony formation assay and quantified by SRB kit at 14d post-XRT. Apoptosis was measured at 72 and 96h post-XRT, and expression of apoptotic proteins (i.e., cleaved PARP and cleaved CASPASE 3) was examined by immunoblotting. (iii) To determine radiosensitivity in vivo, we randomized 20 nude mice bearing QGP-1 xenografts into four groups: control; 3-AP (10mg/Kg); XRT (2 Gy); and combination (XRT immediately followed by 3-AP); each mouse received 5 consecutive treatments. Results: (i) A dose of 2500nM 3-AP arrested cells before entering G2/M phase, significantly reducing percentage of G2/M cells from 8.4% to 0.5% at 72h. Concurrently, we observed increased expression of checkpoint proteins (pRb, pCdk1, pCdk2, pCdc2) and decreased expression of cell cycle inhibitory proteins (cyclin D1, p21, p27 Kip). (ii) Exposure of QGP-1 and BON cells to 3-AP prior to XRT resulted in a synergistic decrease (i.e., 85% and 90%, respectively) in clonogenicity. Furthermore, the combination of 3-AP and XRT increased apoptosis 5-fold and cleaved PARP expression in BON cells. (iii) A significant decrease in tumor size was noted in mice with XRT alone compared to control (146mm2 vs. 280 mm2, respectively); combination treatment (3-AP + XRT) demonstrated a further 48% decrease in tumor size compared to XRT alone. There was no significant decrease in tumor size with 3-AP treatment alone. Conclusion: 3-AP treatment prior to XRT therapy resulted in significant increase in apoptosis in vitro and decrease in tumor size in vivo, suggesting synergistic cytotoxicity and radiosensitivity. Selective RNR inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs.
Citation Format: Zeta Chow, Jeremy Johnson, Aman Chauhan, Tadahide Isumi, Lowell Anthony, Courtney M. Townsend, B. Mark Evers, Piotr Rychahou. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors abstract. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-014.
Abundant studies have associated colorectal cancer (CRC) treatment delay with advanced diagnosis and worse mortality. Delay in seeking specialist is a contributor to CRC treatment delay. The goal of ...this study is to investigate contributing factors to 14-d delay from diagnosis of CRC on colonoscopy to the first specialist visit in the state of Kentucky.
The Kentucky Cancer Registry (KCR) database linked with health administrative claims data was queried to include adult patients diagnosed with stage I-IV CRC from January 2007 to December 2012. The dates of the last colonoscopy and the first specialist visit were identified through the claims. Bivariate and logistic regression analysis was performed to identify factors associated with delay to CRC specialist visit.
A total of 3927 patients from 100 hospitals in Kentucky were included. Approximately, 19% of patients with CRC visited a specialist more than 14 d after CRC detection on colonoscopy. Delay to specialist (DTS) was found more likely in patients with Medicaid insurance (OR 3.1, P < 0.0001), low and moderate education level (OR 1.4 and 1.3, respectively, P = 0.0127), and stage I CRC (OR 1.5, P < 0.0001). There was a higher percentage of delay to specialist among Medicaid patients (44.0%) than Medicare (18.0%) and privately insured patients (18.8%).
We identified Medicaid insurance, low education attainment, and early stage CRC diagnosis as independent risk factors associated with 14-d delay in seeking specialist care after CRC detection on colonoscopy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Kentucky has one of the highest mortality rates for colon cancer, despite dramatic improvements in screening. The National Comprehensive Cancer Network (NCCN) guidelines recommend operation and ...adjuvant chemotherapy for locally advanced (stage IIb/c and stage III) colon cancer (LACC). The purpose of this study was to determine the rate of nonadherence with current standard of care (SOC) and associated factors as possible contributors to mortality.
The Kentucky Cancer Registry database linked with administrative health claims was queried for individuals (20 years and older) diagnosed with LACC from 2007 to 2012. Bivariate and logistic regression of nonadherence was performed. Survival analysis was performed with Cox regression and Kaplan-Meier plots.
A total of 1,404 patients with LACC were included. Approximately 42% of patients with LACC were noted to be nonadherent to SOC, with nearly all (95.7%) failing to receive adjuvant chemotherapy. After adjusting for all significant factors, we found the factors associated with nonadherence included the following: age older than 75 years, stage III colon cancer, high Charlson Comorbidity Index (3+), low poverty level, Medicaid coverage, and disability. Adherence to SOC is associated with a significant improvement in the 5-year survival rate compared with nonadherence (63.0% and 27.4%, respectively; p < 0.0001).
Our study identified multiple factors associated with the failure of patients with LACC to receive SOC, particularly adjuvant chemotherapy, suggesting the need to focus on improving adjuvant chemotherapy compliance in specific populations. Nonadherence to LACC SOC is likely a major contributor to the persistently high mortality rates in Kentucky.
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