Human Cancer Therapy Research Articles

New perspectives for targeting therapy in ALK-positive human cancers.

Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor protein-tyrosine kinase superfamily and was first discovered in anaplastic large-cell lymphoma (ALCL). ALK alterations, including fusions, over-expression and mutations, are highly associated with cancer initiation and progression. This kinase plays an important role in different cancers, from very rare to the more prevalent non-small cell lung cancers. Several ALK inhibitors have been developed and received Food and Drug Administration (FDA) approval. However, like other drugs used in targeted therapies, ALK inhibitors inevitably encounter cancer cell resistance. Therefore, monoclonal antibody screening based on extracellular domain or combination therapies may provide viable alternatives for treating ALK-positive tumors. In this review, we discuss the current understanding of wild-type ALK and fusion protein structures, the pathological functions of ALK, ALK target therapy, drug resistance and future therapeutic directions.

A multifunctional nanoplatform based on Fe3O4@Au NCs with magnetic targeting ability for single NIR light-triggered PTT/PDT synergistic therapy of cancer

Although simultaneous photothermal therapy (PTT) and photodynamic therapy (PDT) have been proven to be beneficial for cancer treatment, high-power laser light sources, shallow penetration depth of light and especially activation by two different light sources make it difficult to be popularized in clinical cancer therapy. Herein, the novel multifunctional Fe3O4@Au nanocomposites (NCs) have been designed and synthesized, which simultaneously act as photothermal conversion agents (PCAs) and photosensitizers (PSs) for PTT/PDT synergistic therapy of human cervical cancer (HeLa) cells using a single excitation light source. Under low-power 808 nm laser irradiation, photothermal conversion efficiency (PCE) of Fe3O4@Au NCs was as high as 39.22% and the cytotoxic singlet oxygen (1O2) could be simultaneously generated. In addition, Fe3O4@Au NCs showed relatively good biocompatibility and low cytotoxicity, which were ideal alternatives for nanotherapeutic agents for treatment of HeLa cells. Meanwhile, PTT only and PTT/PDT dual-modal therapeutic effects of Fe3O4@Au NCs on HeLa cells were compared. The experimental results confirmed that the near-infrared (NIR)-triggered PDT/PTT synergistic therapy of HeLa cells was more efficient due to the synergistic effect. Interestingly, Fe3O4@Au NCs with saturation magnetization (Ms) value of 66.5 emu/g possessed good magnetic responsivity, which could be collected easily with a magnet. The magnetic targeting of Fe3O4@Au NCs could effectively promote their accumulation at tumor sites, thus further enhancing the efficacy of PTT/PDT synergistic therapy of HeLa cells. This study provides a feasible paradigm for magnetic targeting-assisted PTT/PDT synergistic therapy of cancer cells under a single-wavelength excitation and opens up a novel direction for clinical cancer therapy.

Discovery of novel BTK PROTACs with improved metabolic stability via linker rigidification strategy

Bruton's Tyrosine Kinase (BTK) functions as a key regulator of B-cell receptor (BCR) signaling pathway, which is frequently hyperactivated in a variety of lymphoma cancers. Using Proteolysis Targeting Chimera (PROTAC) technology, we have recently discovered a highly potent ARQ-531-derived BTK PROTAC 6e, inducing effective degradation of both wild type (WT) and C481S mutant BTK proteins. However, the poor metabolic stability of PROTAC 6e have limited its further in vivo studies. Herein, we present our structure-activity relationship (SAR) studies on modifying PROTAC 6e using linker rigidification strategy to identify a novel cereblon (CRBN)-recruiting compound 3e that induced BTK degradation in a concentration-dependent manner but had no effect on reducing the level of CRBN neo-substrates. Moreover, compound 3e suppressed the cell growth more potently than the small molecule inhibitors ibrutinib and ARQ-531 in several cells. Furthermore, compound 3e with the rigid linker displayed a significantly improved metabolic stability profile with the T1/2 increased to more than 145 min. Overall, we discovered a highly potent and selective BTK PROTAC lead compound 3e, which could be further optimized as potential BTK degradation therapy for BTK-associated human cancers and diseases.

Abstract 408: Novel growth hormone receptor antagonist with discrete PEGylations markedly improve response to therapy in human pancreatic and bile duct cancers in mice

Abstract Here we present a new and improved GHR antagonist candidate (compound G) with novel discrete PEGylations exhibiting marked efficacy in synergizing anti-cancer therapies. The American Cancer Society estimates the 5-year survival rates of pancreatic adenocarcinoma (PAAD) and bile duct cancer/cholangiocarcinoma (CHOL) to be a dismal 10% in 2022, indicating an urgent need for new therapeutic approaches for both of these devastating diseases. Several human cancers, including PAAD and CHOL express two validated oncological targets - the growth hormone receptor (GHR) and the insulin-like growth factor 1 receptor (IGF1R), as well as their cognate ligands growth hormone (GH) and insulin-like growth factor 1 (IGF1), respectively. Our laboratory had pioneered the discovery of pharmacologic antagonists for the growth hormone receptor (GHR), including the only approved and prescribed GHR antagonist, pegvisomant (Pfizer), prescribed for acromegaly (a condition of excess GH due to a hypersecreting pituitary adenoma). GHR antagonists not only inhibit the effects of GH but also robustly suppress the production of IGF1. The novel GHR antagonist compound G, that we present here, has a greater binding affinity than Pegvisomant to the GHR. And unlike Pegvisomant, compound G binds to the prolactin receptor as well as the GHR. A large body of research by us and others have earlier shown that the covert actions of tumoral GHR and IGF1R activation collectively drive mechanisms of therapy resistance including, but not limited to, active multidrug efflux, activation of epithelial-to-mesenchymal transition (EMT), resistance to apoptosis, increased fibrosis, increase of cancer stem cells, tumor angiogenesis and senescence. Our novel discrete PEGylated GHR antagonist, compound G, alone exhibits significant suppression of cancer cell viability, invasive potential, and drug efflux rate in PAAD and CHOL cell cultures. In Nude mice with human PAAD or CHOL xenografts, GHR inhibition by compound-G markedly sensitized the tumors towards both low and high doses of gemcitabine (for PAAD) or gemcitabine-cisplatin (for CHOL) treatments. Compound-G in combination with chemotherapy resulted in markedly reduced tumor sizes in all CHOL and PAAD mice and almost complete tumor clearance in 40% of the mice with PAAD. In the GHR antagonist treated tumors, significantly lower expression of ABC transporters (Abcb1, Abcg5, Abcc2, Abcg2), EMT mediators (Cdh2, Zeb1, Snai2, Epas1) and apoptosis marker (Bcl2) genes compared to control were observed - accounting for the molecular mechanisms underlying the observed effects. Collectively, our data provide a robust pre-clinical validation for treatment regimens combining GHR antagonist and chemotherapy leading to highly efficacious tumor clearance. Citation Format: Reetobrata Basu, John J. Kopchick. Novel growth hormone receptor antagonist with discrete PEGylations markedly improve response to therapy in human pancreatic and bile duct cancers in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 408.

Abstract 687: Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis

Abstract ES2B-C001 is a virus-like particle (VLP) vaccine against human HER-2, developed for the therapy of breast cancer. We show here that ES2B-C001 effectively inhibits mammary carcinoma growth and metastasis in a transgenic mouse model expressing activated human HER-2. ES2B-C001 vaccine is a tag/catcher conjugation system: Acinectobacter phage 205 (AP205) capsid VLP, each with 180 tag peptides, are conjugated with catcher-HER-2 extracellular domain. The vaccine was administered alone, thanks to the intrinsic adjuvanticity of the VLP, or with Montanide ISA 51. QD is a HER-2-positive cell line established from a mammary carcinoma of a Delta16 (FVB background) transgenic mouse, bearing the human HER-2 splice variant Delta16. FVB female mice were challenged in the mammary fat pad with 1 million QD cells; vaccinations started 2 weeks after cell challenge and were repeated every 2 weeks. Untreated mice developed progressive tumors within one month, whereas 70% of mice vaccinated without adjuvant and all mice vaccinated with adjuvant were still tumor-free one year after cell challenge. Mice challenged intravenously (i.v.) developed more than 300 lung metastases, whereas all vaccinated mice remained metastasis-free. Delta16 transgenic mice are immunologically tolerant to human HER-2 and develop aggressive mammary carcinomas with a median latency of 5 months. Vaccination of young, tumor-free Delta16 mice completely prevented tumor onset for more than one year. Delta16 mice challenged i.v. with QD cells mice developed a mean of 68 lung nodules, whereas 73% of mice therapeutically vaccinated without adjuvant, and all mice vaccinated with E2SB-C001+ISA 51, were free from metastases. ES2B-C001 induced copious anti-HER-2 antibodies of all IgG subclasses, ranging 1-10 mg/mL in FVB mice and 0.1-1 mg/mL in Delta16 mice; antibody titers remained very high for 6-10 months after the last vaccination. Antibodies inhibited the 3D growth of human HER-2+++ and HER-2++ breast cancer cells, of trastuzumab resistant cells and of gastric carcinoma cells. Vaccination increased interferon-gamma secreting cells in the spleen, as evaluated by ELISPot (21±3 spots/2x105 cells). The results warrant further development of ES2B-C001 for the therapy of HER-2 positive human breast cancer and of other tumors expressing HER-2. Citation Format: Francesca Ruzzi, Arianna Palladini, Stine Clemmensen, Annette Strobaek, Nicolaas Buijs, Tanja Domeyer, Jerzy Dorosz, Vladislav Soroka, Dagmara Grzadziela, Christina Jo Rasmussen, Ida Busch Nielsen, Max Soegaard, Maria Sofia Semprini, Laura Scalambra, Stefania Angelicola, Lorena Landuzzi, Pier-Luigi Lollini, Mette Thorn. Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 687.

Anticancer Activity of Region B Capsaicin Analogs.

The heterocyclic vanilloid compound capsaicin is responsible for the spicy and pungent flavor of chili peppers. Several convergent studies have shown that capsaicin suppresses the growth of multiple human cancers. Apart from capsaicin, natural and synthetic capsaicin-like compounds display growth suppressive activity in human cancers. The pharmacophore of capsaicin is comprised of three regions, namely region A (the aromatic ring), region B (the amide bond), and region C (the side chain). The present manuscript describes the isolation and synthesis of capsaicin analogs which have structural modifications in region B of the molecule. Furthermore, the pharmacokinetic properties, anticancer activity of region B capsaicin analogs, as well as the signaling pathways (underlying the growth-inhibitory effects of region B capsaicin analogs) have also been described. The discovery of novel, second-generation region B capsaicin analogs may foster the hope of innovative nutrition-based combination therapies in human cancers.

Total Flavone Content of Cydonia oblonga Miller Inhibits Proliferation and Migration of Renal Carcinoma Cells by Inhibiting the S1PR2/FAK Pathway

Cydonia oblonga Miller (C. oblonga) is known for its beneficial properties for health since ancient times. C. oblonga has also been shown to possess antihemolytic, antidiabetic, and antilipoperoxidation, as well as lipid-lowering properties. Modern research has shown that C. oblonga also possess antitumor effects. However, studies have not reported whether its main compounds inhibit renal cell carcinoma (RCC) development and progression. We found the inhibitory effect of total flavone of C. oblonga (TFCOM) in RCC cells through in vitro screening tests. The molecular mechanism of this effect is still unknown. Therefore, molecular docking combined with network pharmacological methods was used in order to clarify the molecular mechanisms of TFCOM anticancer effects. TFCOM showed potent inhibitory effects on the proliferation and migration of 786-O and Renca cells. Hoechst 33342 staining test results indicated that TFCOM’s cell growth inhibition in RCC cells may be mediated through apoptosis. It may significantly influence S1PR2/FAK, WNT/β-catenin, and PI3K/AKT/mTOR signaling pathways that can regulate a series of cellular activities and can participate in the processes of cell proliferation, migration, and apoptosis. We conclude that TFCOM has antitumor activity against kidney cancer. It may induce apoptosis and inhibitory effects on cell proliferation and migration through S1PR2/FAK, WNT/β-catenin, and PI3K/AKT/mTOR signal pathways. TFCOM might be a potential anticancer drug to be further developed for human kidney cancer therapy. In recent years, many synthetic drugs with anticancer effects have the disadvantages of high price and side effects. Thus, the development of anticancer drugs from natural resources has its application value. Our results suggest that TFCOM, as a natural product, has multiple target effects, and it may act through proteins and receptors such as FAK and S1PR2, thereby affecting the activation of downstream proteins and inhibiting the occurrence and development of renal cancer cells.

Targeted Two-Step Delivery of Oncotheranostic Nano-PLGA for HER2-Positive Tumor Imaging and Therapy In Vivo: Improved Effectiveness Compared to One-Step Strategy

Therapy for aggressive metastatic breast cancer remains a great challenge for modern biomedicine. Biocompatible polymer nanoparticles have been successfully used in clinic and are seen as a potential solution. Specifically, researchers are exploring the development of chemotherapeutic nanoagents targeting the membrane-associated receptors of cancer cells, such as HER2. However, there are no targeting nanomedications that have been approved for human cancer therapy. Novel strategies are being developed to alter the architecture of agents and optimize their systemic administration. Here, we describe a combination of these approaches, namely, the design of a targeted polymer nanocarrier and a method for its systemic delivery to the tumor site. Namely, PLGA nanocapsules loaded with a diagnostic dye, Nile Blue, and a chemotherapeutic compound, doxorubicin, are used for two-step targeted delivery using the concept of tumor pre-targeting through the barnase/barstar protein "bacterial superglue". The first pre-targeting component consists of an anti-HER2 scaffold protein, DARPin9_29 fused with barstar, Bs-DARPin9_29, and the second component comprises chemotherapeutic PLGA nanocapsules conjugated to barnase, PLGA-Bn. The efficacy of this system was evaluated in vivo. To this aim, we developed an immunocompetent BALB/c mouse tumor model with a stable expression of human HER2 oncomarkers to test the potential of two-step delivery of oncotheranostic nano-PLGA. In vitro and ex vivo studies confirmed HER2 receptor stable expression in the tumor, making it a feasible tool for HER2-targeted drug evaluation. We demonstrated that two-step delivery was more effective than one-step delivery for both imaging and tumor therapy: two-step delivery had higher imaging capabilities than one-step and a tumor growth inhibition of 94.9% in comparison to 68.4% for the one-step strategy. The barnase*barstar protein pair has been proven to possess excellent biocompatibility, as evidenced by the successful completion of biosafety tests assessing immunogenicity and hemotoxicity. This renders the protein pair a highly versatile tool for pre-targeting tumors with various molecular profiles, thereby enabling the development of personalized medicine.

Abstract P2-20-08: Expression of ACVRL1 in a subset of recruited tumor-associated macrophages drives resistance to PD-1 therapy in human breast cancer

Abstract The activin-like kinase receptor 1 (ALK1, encoded by ACVRL1) is a member of the transforming growth factor β (TGF-β) superfamily. Expression of ALK1 has been historically associated with endothelial cells, thus serving as a potential target for antiangiogenic therapy in cancer. Despite promising preclinical results with a ligand trap (RAP-041/dalantercept), clinical trials did not show additional benefit of dalantercept when combined or contrasted with the standard of care. This outcome raises the question about patient selection and optimal combination partner in the clinical setting. Moreover, from the biological perspective, how ALK1 signaling shapes the features of a tumor is still largely uncharted. Based on our previous observations, we confirmed that the genetic network correlated with ACVRL1 expression is associated with immune processes in human breast cancer. Next, we validated that inhibition of ALK1 altered the immune landscape in experimental primary breast cancer (MMTV-PyMT model), providing a rationale for a combined regimen with immunotherapy. After developing and validating a murine model of adjuvant therapy with RAP-041 (syngeneic E0771 and 4T1 cell lines), we discovered that ALK1 blockade potentiated immune checkpoint inhibitors. Based on bulk RNA-sequencing and a multicolor FACS approach, addition of RAP-041 to immunotherapy significantly reduced the number of intratumoral macrophages compared with either treatment alone. Notably, these changes were coupled with systemic alterations in peripheral blood cell composition, with the combined administration suppressing circulating monocytes. Interestingly, ALK1 signaling appears to be a pivotal contributor to this phenotype, as fewer circulating monocytes were also detected in the RAP-041 cohort. Based on our in vitro characterization, we revealed that ALK1 expression is not limited to endothelial cells, as different myeloid populations readily expressed Acvrl1, suggesting a direct functional role of ALK1. Stimulation of ALK1 did not alter macrophage polarization, even though different levels of mediators downstream of ALK1 characterized the M1/M2 states. Furthermore, apoptosis and necrosis did not seem to be affected upon activation of ALK1, while the effect on the cell cycle awaits confirmation. In vitro genetic modulation of ALK1 activity is ongoing, with co-culture systems, transendothelial adhesion and migration being currently assessed. Inspection of several human sc-RNA-sequencing datasets confirmed the existence of ACVRL1-positive monocytes in human PBMCs from healthy individuals as well as in human solid cancers, including breast cancer. Focusing on the immune compartment, ACVRL1 expression characterized recruited tumor-associated macrophages (TAMs) and their cycling counterpart. In light of this feature, longitudinal follow-up of patients exposed to chemotherapy and/or anti-PD-1/PD-L1 revealed that expression of ACVRL1 decreased following exposure to chemotherapy. Notably, high ACVRL1 expression and an increased proportion of ACVRL1-positive TAMs were found at progression with anti-PD-1 treatment. In agreement with our preclinical data, ACVRL1-positive cells were depleted from circulation upon recurrence, indicating a migration from blood to tumors. Finally, survival analysis determined that ACVRL1hi signature in macrophages correlated with a significantly lower survival in a 5-year follow-up in breast cancer. In conclusion, our work sheds light on the value of ALK1 as a promising dual antiangiogenic and immunomodulatory target for precision medicine in cancer. Citation Format: Mehrnaz Safaee Talkhoncheh, Jonas Sjölund, Paulina Bolivar Balbas, Ewa Kurzejamska, Sara Larsson, Eugenia Cordero, Clara R. Oudenaarden, Jessica Pantaleo, Göran Jonsson, Charlotte Rolny, Matteo Bocci, Kristian Pietras. Expression of ACVRL1 in a subset of recruited tumor-associated macrophages drives resistance to PD-1 therapy in human breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-08.

Abstract OT2-14-01: Pyrotinib after trastuzumab-based adjuvant therapy in high-risk early human epidermal growth factor receptor 2-positive breast cancer: a multicenter phase 2 trial

Abstract Objective: Trastuzumab-based adjuvant therapy was demonstrated to improve the prognosis of early human epidermal growth factor receptor 2 (HER-2) positive breast cancer, while about 10% of patients showed primary resistance. ExteNET study showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, after trastuzumab-based adjuvant therapy significantly improved invasive disease-free survival (iDFS) in women with HER2-positive breast cancer. Besides, for patients with early hormone receptor positive breast cancer within 1 year of prior trastuzumab with residual disease after neoadjuvant therapy, extended adjuvant neratinib showed prolonged overall survival, which suggested the potential benefit of extended adjuvant therapy for high-risk patients. Pyrotinib is an oral irreversible pan-HER receptor tyrosine kinase inhibitor targeting epidermal growth factor receptor, HER2, and HER4, which showed promising efficacy and manageable safety profiles in the treatment of HER-2 positive advanced breast cancer. In this study, we aimed to evaluate the efficacy and safety of extended adjuvant pyrotinib following trastuzumab-based adjuvant therapy in patients with high-risk early HER-2 positive breast cancer. Methods: In this ongoing multicenter, open-label, phase II trial at 23 centers in China, women with operable high-risk early HER-2 positive breast cancer and known hormone receptor status, who have completed trastuzumab-based adjuvant therapy within 6 months were enrolled. High-risk patients must meet one of the following criteria: N stage ≥1; T stage ≥2; did not achieve pathological complete response (pCR) after neoadjuvant therapy; had pCR after neoadjuvant therapy but with tumor size ≥ 5cm or N stage ≥2; or tumor size less than 2cm but with high Ki67, histologic grade 3 or with lymph node micrometastasis. Eligible patients were assigned to receive 6 month or 1 year of oral pyrotinib 400mg/day. The primary endpoint was the 2-year iDFS rate. Results: Between January, 2019 and February, 2022, a total of 142 eligible women were enrolled and assigned to receive pyrotinib, with a median age of 50 (range: 25-72) years old. Ninety-seven patients had a T stage ≥2. A total of 108 patients (76.1%) had node-positive disease, and 64 (45.1%) were hormone receptor positive. Twenty-five patients used neoadjuvant therapy previously, and all of them did not receive pCR (Table 1). After a median follow-up of 20 (range: 0.25-41) months, the 2-year iDFS rate was not available. Two and four patients reported invasive disease events after 6-month and 1-year of follow-up, with the 6-month and 1-year iDFS rates of 98.6% and 97.2%, respectively. The most common adverse events reported was diarrhea (78.2%), followed by fatigue (36.6%), lymphocyte count decreased (36.6%), nausea (33.1%) and hand-foot syndrome (33.1%). Forty-three patients experienced grade 3 diarrhea, and no grade 4 or higher adverse event was reported (Table 2). Adverse event leading to treatment discontinuation occurred in 12 (8.5%) patients. Conclusion: Extended adjuvant pyrotinib administrated after trastuzumab-based adjuvant therapy was well-tolerated and showed potential efficacy in high-risk early HER-2 positive breast cancer. The follow-up is ongoing to determine the long-term benefit of extended adjuvant pyrotinib. Table 1. Baseline characteristics of patients ER: estrogen receptor; PR: progesterone receptor. Table 2. Treatment-emergent adverse event occurring in at least 10% of the patients Citation Format: Feilin Cao, Zhaosheng Ma, Guinv Hu, Xiaotao Zhu, Shuguang Li, Shuzheng Chen, Binglie Chen, Zhanwen Li, Weizhu Wu, Xiaochun Ji, Jingde Shu, Deyou Tao, Xiaoqing Hu, Min Zheng, Ouchen Wang, Qingjing Feng, Jing Hao, Xujun Li. Pyrotinib after trastuzumab-based adjuvant therapy in high-risk early human epidermal growth factor receptor 2-positive breast cancer: a multicenter phase 2 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-14-01.

Efficacy of resveratrol against breast cancer and hepatocellular carcinoma cell lines.

To evaluate the anti-cancer effect of resveratrol on Michigan cancer foundation-7 (MCF-7) and hepatoblastoma cell line (HepG2) cells. The study was carried out at the Department of Botany and Microbiology, Prince Sattam bin Abdulaziz University, Al-kharj, Saudi Arabia, from August 2022 to October 2022. Different concentrations of resveratrol were added to the MCF-7 and HepG2 cell lines. Cell death and proliferation were measured with MTT and Trypan blue exclusion assays. Apoptosis markers were assessed by using a quantitative PCR assay (qPCR). The resveratrol was shown to suppress the proliferation of MCF-7 and HepG2 cells at dose- and time-dependent. The cytotoxic effect of resveratrol was observed even at 100 μM after 24 hours. In comparison to untreated cells, resveratrol treatment reduced the viability of MCF-7 cells to roughly 57.5% with a half maximal inhibitory concentration (IC50) of 51.18 μM and HepG2 cells to 56.2% with an IC50 of 57.4 μM. Furthermore, in the tested cell lines, resveratrol was able to induce apoptosis mediated by elevated apoptosis markers. Resveratrol appears to be an excellent candidate agent in anticancer therapy in various human cancers.

Abstract P3-07-26: Anti-cancer activity of naturally formulated Trigonella foenum graecum (Fenugreek) PLGA hyaluronic acid-coated nanoparticles for treatment of breast cancer cell lines

Abstract There are several growing evidences of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat several disorders such as diabetes, inflammation, and gastrointestinal ailments. Previously, we reported that Fenugreek has anticancer properties against liver cancer due to its active beneficial chemical constituents. Herein, Fenugreek-loaded poly (l-lactic-co-glycolic acid) (PLGA) hyaluronic acid (HA)-coated nanoparticles have been formulated to improve and enhance the anticancer potential of Fenugreek against human breast cancer cell lines. The cytotoxic effects, physicochemical characterization in vitro and pharmacodynamic study of optimized formulation of the FCE-loaded (PLGA)-(HA)-coated nanoparticles in vivo were investigated in (ER-positive) and (triple-negative), breast cancer cell lines, ZR-75-1 and MDA-MB-231, respectively. The particle size, PDI, zeta potential, entrapment efficiency, and loading capacity of FCE-loaded nanoparticles were 188.5 ± 3.1 d.nm, 0.198 ± 0.114, −.118 mV, 38.41 ± 1.4%, and 6.37 ± 1.08%, respectively. The in vitro cytotoxicity study of FCE-loaded NPs by Wst-1 proliferation assay on both tested breast cancer cell lines showed an anti-cancer activity after 48 h of treatment in a dose-dependent manner. Furthermore, the anticancer effect of FCE-loaded nanoparticles was examined by the morphological cell changes, histone release, caspase-3 activity, and modulation of the oncogenic signal pathways of m-tor, β-catenin, cyclin D-1 and STAT3 that are constitutively active in wide variety of breast cancer cell lines which in turn could be important as therapeutic targets in breast cancer. 66 μg/mL FCE-loaded PLGA nanoparticles induced 67.3% apoptosis in both tested cell lines after 48 h that was associated with increased expression of caspase 3, Bax, Bak, p53, and cell cycle arrest at G0/G1 in a time-and dose-dependent manner compared to unloaded-nanoparticles or untreated cells. Moreover, there were significant inhibition in m-tor, β-catenin, cycline-D1, and STAT3 expression levels in both tested cell lines compared to un-loaded-nanoparticles or untreated cells. All in all, our results showed that the formulated FCE-loaded (PLGA)-(HA)-coated nanoparticles induced apoptosis in both ER-positive and triple-negative breast cancer cell lines respectively by increase the expression of pro-apoptotic genes and inhibited several oncogenic genes that might hold a promise for the complementary therapy for human breast cancer. Citation Format: Islam Yahiya, Rana Saeed, Ahmed Sultan. Anti-cancer activity of naturally formulated Trigonella foenum graecum (Fenugreek) PLGA hyaluronic acid-coated nanoparticles for treatment of breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-26.

The Role of Exosomes in Human Carcinogenesis and Cancer Therapy-Recent Findings from Molecular and Clinical Research.

Exosomes are biological nanoscale spherical lipid bilayer vesicles, 40-160 nm in diameter, produced by most mammalian cells in both physiological and pathological conditions. Exosomes are formed via the endosomal sorting complex required for transport (ESCRT). The primary function of exosomes is mediating cell-to-cell communication. In terms of cancer, exosomes play important roles as mediators of intercellular communication, leading to tumor progression. Moreover, they can serve as biomarkers for cancer detection and progression. Therefore, their utilization in cancer therapies has been suggested, either as drug delivery carriers or as a diagnostic tool. However, exosomes were also reported to be involved in cancer drug resistance via transferring information of drug resistance to sensitive cells. It is important to consider the current knowledge regarding the role of exosomes in cancer, drug resistance, cancer therapies, and their clinical application in cancer therapies.

The E3 ubiquitin ligases regulate PD-1/PD-L1 protein levels in tumor microenvironment to improve immunotherapy.

The tumor microenvironment (TME) is the tumor surrounding environment, which is critical for tumor development and progression. TME is also involved in clinical intervention and treatment outcomes. Modulation of TME is useful for improving therapy strategies. PD-L1 protein on tumor cells interacts with PD-1 protein on T cells, contributing to T cell dysfunction and exhaustion, blockage of the immune response. Evidence has demonstrated that the expression of PD-1/PD-L1 is associated with clinical response to anti-PD-1/PD-L1 therapy in cancer patients. It is important to discuss the regulatory machinery how PD-1/PD-L1 protein is finely regulated in tumor cells. In recent years, studies have demonstrated that PD-1/PD-L1 expression was governed by various E3 ubiquitin ligases in TME, contributing to resistance of anti-PD-1/PD-L1 therapy in human cancers. In this review, we will discuss the role and molecular mechanisms of E3 ligases-mediated regulation of PD-1 and PD-L1 in TME. Moreover, we will describe how E3 ligases-involved PD-1/PD-L1 regulation alters anti-PD-1/PD-L1 efficacy. Altogether, targeting E3 ubiquitin ligases to control the PD-1/PD-L1 protein levels could be a potential strategy to potentiate immunotherapeutic effects in cancer patients.

Immune Checkpoint Molecule TIGIT Regulates Kidney T Cell Functions and Contributes to AKI.

T cells mediate pathogenic and reparative processes during AKI, but the exact mechanisms regulating kidney T cell functions are unclear. This study identified upregulation of the novel immune checkpoint molecule, TIGIT, on mouse and human kidney T cells after AKI. TIGIT-expressing kidney T cells produced proinflammatory cytokines and had effector (EM) and central memory (CM) phenotypes. TIGIT-deficient mice had protection from both ischemic and nephrotoxic AKI. Single-cell RNA sequencing led to the discovery of possible downstream targets of TIGIT. TIGIT mediates AKI pathophysiology, is a promising novel target for AKI therapy, and is being increasingly studied in human cancer therapy trials. T cells play pathogenic and reparative roles during AKI. However, mechanisms regulating T cell responses are relatively unknown. We investigated the roles of the novel immune checkpoint molecule T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in kidney T cells and AKI outcomes. TIGIT expression and functional effects were evaluated in mouse kidney T cells using RNA sequencing (RNA-Seq) and flow cytometry. TIGIT effect on AKI outcomes was studied with TIGIT knockout (TIGIT-KO) mice in ischemia reperfusion (IR) and cisplatin AKI models. Human kidney T cells from nephrectomy samples and single cell RNA sequencing (scRNA-Seq) data from the Kidney Precision Medicine Project were used to assess TIGIT's role in humans. RNA-Seq and flow cytometry analysis of mouse kidney CD4+ T cells revealed increased expression of TIGIT after IR injury. Ischemic injury also increased TIGIT expression in human kidney T cells, and TIGIT expression was restricted to T/natural killer cell subsets in patients with AKI. TIGIT-expressing kidney T cells in wild type (WT) mice had an effector/central memory phenotype and proinflammatory profile at baseline and post-IR. Kidney regulatory T cells were predominantly TIGIT+ and significantly reduced post-IR. TIGIT-KO mice had significantly reduced kidney injury after IR and nephrotoxic injury compared with WT mice. scRNA-Seq analysis showed enrichment of genes related to oxidative phosphorylation and mTORC1 signaling in Th17 cells from TIGIT-KO mice. TIGIT expression increases in mouse and human kidney T cells during AKI, worsens AKI outcomes, and is a novel therapeutic target for AKI.

Cross-reactivity of anti-human programmed cell death ligand 1 (PD-L1) monoclonal antibody, clone 28-8 against feline PD-L1.

Immunotherapy is a breakthrough in human cancer therapy and has become a major concern in veterinary oncology. However, in cats, many unclear points of the tumor microenvironment exist, including immune checkpoint molecules. A reason is that very few monoclonal antibodies have been proven to react with feline molecules. Therefore, this study investigated whether anti-human programmed cell death ligand 1 (PD-L1) monoclonal antibody, clone 28-8, which is currently commercially available, can also recognize feline PD-L1 by flow cytometry, immunoprecipitation, and immunohistochemical (IHC) staining. We confirmed that the antibody's specificity by flow cytometry and immunoprecipitation using NIH3T3 cells transfected with feline PD-L1. Additionally, we revealed that PD-L1 was expressed on the surface of some feline cell lines by flow cytometry and clone 28-8 antibody unbound to the cells where feline PD-L1 was knocked out. Furthermore, IHC analysis revealed that PD-L1 was expressed in macrophages in the spleen and lymph nodes from healthy cats and mast cell tumor cells. Therefore, we indicated that the clone 28-8 antibody is a valuable tool in detecting feline PD-L1, and further analysis of tumor tissues is expected in the future.

Inhibition of Cytochrome P450 by Carbon Monoxide: Relevance to Drug Resistance in Human Breast Cancer Therapy

Drug resistance to conventional chemotherapeutics is a great impediment to cancer therapy. A major part of this problem arises from rapid metabolism of the drugs by cytochrome P450 class of enzymes before they reach their targets or at the target itself. Inhibition of such enzymatic deactivation of the drugs could offer partial rescue and make chemotherapy more effective. Site specific delivery of exogenous carbon monoxide has been shown to inhibit cytochrome P450 enzymes and resurrect sensitivity to chemotherapeutics already available in the market. Successful design for application of such CO delivery will thus be extremely desirable to patients particularly in poor countries where the antibody-based or nanodrug therapies, discovered recently, are too expensive for the general population. The potential of such carbon monoxide-induced cytochrome P450 inhibition to improve drug sensitization to conventional chemotherapeutics in breast cancer therapy has been discussed in this account.

Timosaponin AIII Inhibits Migration and Invasion Abilities in Human Cervical Cancer Cells through Inactivation of p38 MAPK-Mediated uPA Expression In Vitro and In Vivo

Cervical cancer is one of the most common gynecologic cancers globally that require novel approaches. Timosaponin AIII (TSAIII) is a steroidal saponin that displays beneficial effects in antitumor activities. However, the effect of TSAIII on human cervical cancer remains unknown. In this study, we found that TSAIII showed no influence on cell viability, cytotoxicity, cell cycle distribution and apoptosis induction in human cervical cancer cells. TSAIII was revealed to have a significant inhibitory effect on cell migration and invasion through the downregulation of invasion-related uPA expression and p38 MAPK activation in both human cervical cancer cells and cervical cancer stem cells (CCSCs), indicating that the p38 MAPK-uPA axis mediated the TSAIII-inhibited capacity of cellular migration and invasion. In a synergistic inhibition assay, a TSAIII plus p38 siRNA cotreatment revealed a greater inhibition of uPA expression, migration and invasion in human cervical cancer cells. In an immunodeficient mouse model, TSAIII significantly inhibited lung metastases from human cervical cancer SiHa cells without TSAIII-induced toxicity. These findings first revealed the inhibitory effects of TSAIII on the progression of human cervical cancer through its downregulation of p38 MAPK-uPA axis activation. Therefore, TSAIII might provide a potential strategy for auxiliary therapy in human cervical cancer.

Abstract A021: Clinical and molecular characterization of naturally-occurring colorectal cancer in rhesus macaques reveals mismatch repair deficiency driven by epigenetic silencing of MLH1

Abstract Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and emerge at similar life stages as in human patients, but the carcinogenesis and molecular background remain elusive, limiting the value of the model for human disease and cancer therapy. In order to better understand this naturally occurring model, we established a diagnostic and staging pipeline including imaging (18F-FDG PET, plain and contrast-enhanced CT), histopathology, and clinical assessments. We observed cancer symptoms and co-morbidities in our cohort (n=16, all Indian-origin, 11 females, mean age at arrival 20.2y) such as hypoalbuminemia, fecal occult blood, and microcytic anemia, as frequently described in human CRC patients. Pathologically, all cancers were right-sided, involving the proximal colon and/or ileocecocolic junction, and most were densely fibrotic, restricting the colonic lumen. Most cancers appeared with glandular morphology and some (18.8%) had mucinous components. Immunohistochemistry revealed loss of MLH1 and PMS2 in 100% of investigated CRCs, indicating mismatch repair deficiency which furthermore resulted in microsatellite instability (PCR & fragment analysis). Whole exome sequencing revealed the close genetic relatedness to human CRCs, particularly exemplified by mutations affecting KRAS (37.5%, e.g., p.G12D), APC (31.3%), TP53 (18.8%, e.g. p.R175H), ARID1A (56.3%), and ALK (43.8%), as similarly annotated in the human COSMIC database. Nonetheless, somatic mutations do not explain the loss of MLH1 in the entire CRC cohort. Transcriptomics on the other hand revealed the transcriptional suppression of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRC in comparison to adjacent healthy colon. Moreover, comparison of differentially expressed gene sets of rhesus CRC with a human annotated database (IPA) confirmed the disease similarities observed clinically, genetically, and histopathologically. Subsequently, we investigated DNA methylation of the promoter region of MLH1 and retranslated markers for the CpG island methylator phenotype (CIMP) as described in human CRC. While only 56.3% of CRCs were considered CIMP positive (≥3/5 markers hypermethylated), 100% of investigated CRCs exhibited MLH1 promoter hypermethylation. As a result, epigenetic silencing is suggested to suppress MLH1 transcription, cause the loss of MLH1 protein, and drive mismatch repair deficiency and genomic instability in naturally occurring CRC in rhesus macaques. We therefore consider spontaneous, uninduced CRC in rhesus macaques, their treatment-naïve nature, and their unaltered immune competence an outstanding model for human disease and in particular for human cancer immunotherapy. Citation Format: Simon Deycmar, Brendan Johnson, Karina Ray, David Caudell Caudell, John Olson, Greg Dugan, W. Shane Sills, Declan Ryan, Christopher Whitlow, Kiran K. Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Cassandra Cullin, Brandy Dozier, Emily Romero, Francois Villinger, Armando Burgos, Jeff Chou, Bruno Gomes, Michael Hettich, Maurizio Ceppi, Jehad Charo, J. Mark Cline. Clinical and molecular characterization of naturally-occurring colorectal cancer in rhesus macaques reveals mismatch repair deficiency driven by epigenetic silencing of MLH1 [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A021.

Induction of Apoptosis via Inactivating PI3K/AKT Pathway in Colorectal Cancer Cells Using Aged Chinese Hakka Stir-Fried Green Tea Extract.

Food extract supplements, with high functional activity and low side effects, play a recognized role in the adjunctive therapy of human colorectal cancer. The present study reported a new functional beverage, which is a type of Chinese Hakka stir-fried green tea (HSGT) aged for several years. The extracts of the lyophilized powder of five HSGT samples with different aging periods were analyzed with high-performance liquid chromatography. The major components of the extract were found to include polyphenols, catechins, amino acids, catechins, gallic acid and caffeine. The tea extracts were also investigated for their therapeutic activity against human colorectal cancer cells, HT-29, an epithelial cell isolated from the primary tumor. The effect of different aging time of the tea on the anticancer potency was compared. Our results showed that, at the cellular level, all the extracts of the aged teas significantly inhibited the proliferation of HT-29 in a concentration-dependent manner. In particular, two samples prepared in 2015 (15Y, aged for 6 years) and 2019 (19Y, aged for 2 years) exhibited the highest inhibition rate for 48 h treatment (cell viability was 50% at 0.2 mg/mL). Further, all the aged tea extracts examined were able to enhance the apoptosis of HT-29 cells (apoptosis rate > 25%) and block the transition of G1/S phase (cell-cycle distribution (CSD) from <20% to >30%) population to G2/M phase (CSD from nearly 30% to nearly 10%) at 0.2 mg/mL for 24 h or 48 h. Western blotting results also showed that the tea extracts inhibited cyclin-dependent kinases 2/4 (CDK2, CDK4) and CylinB1 protein expression, as well as increased poly ADP-ribose polymerase (PRAP) expression and Bcl2-associated X (Bax)/B-cell lymphoma-2 (Bcl2) ratio. In addition, an upstream signal of one of the above proteins, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling, was found to be involved in the regulation, as evidenced by the inhibition of phosphorylated PI3K and AKT by the extracts of the aged tea. Therefore, our study reveals that traditional Chinese aged tea (HSGT) may inhibit colon cancer cell proliferation, cell-cycle progression and promoted apoptosis of colon cancer cells by inactivating PI3K/AKT signalling.