Publications

Weissenrieder, J. S., Weissenkampen, J. D., Reed, J. L., Green, M. V., Zheng, C., Neighbors, J. D., Liu, D. J. & Hohl, R. J., Dec 2022, In: Scientific reports. 12, 1, 359.

The schweinfurthin family of natural compounds exhibit a unique and potent differential cytotoxicity against a number of cancer cell lines and may reduce tumor growth in vivo. In some cell lines, such as SF-295 glioma cells, schweinfurthins elicit cytotoxicity at nanomolar concentrations. However, other cell lines, like A549 lung cancer cells, are resistant to schweinfurthin treatment up to micromolar concentrations. At this time, the precise mechanism of action and target for these compounds is unknown. Here, we employ RNA sequencing of cells treated with 50 nM schweinfurthin analog TTI-3066 for 6 and 24 h to elucidate potential mechanisms and pathways which may contribute to schweinfurthin sensitivity and resistance. The data was analyzed via an interaction model to observe differential behaviors between sensitive SF-295 and resistant A549 cell lines. We show that metabolic and stress-response pathways were differentially regulated in the sensitive SF-295 cell line as compared with the resistant A549 cell line. In contrast, A549 cell had significant alterations in response genes involved in translation and protein metabolism. Overall, there was a significant interaction effect for translational proteins, RNA metabolism, protein metabolism, and metabolic genes. Members of the Hedgehog pathway were differentially regulated in the resistant A549 cell line at both early and late time points, suggesting a potential mechanism of resistance. Indeed, when cotreated with the Smoothened inhibitor cyclopamine, A549 cells became more sensitive to schweinfurthin treatment. This study therefore identifies a key interplay with the Hedgehog pathway that modulates sensitivity to the schweinfurthin class of compounds.

Ruoheng Zhang, J. D. Neighbors, T. D. Schell, R. J. Hohl

Our previous study showed that one of the schweinfurthin compounds, 5’-methoxyschweinfurthin G (MeSG), not only enhances the anti-tumor effect of anti-PD1 antibody in the B16F10 murine melanoma model, but also provokes durable, protective anti-tumor immunity. Here we further investigated the mechanisms by which MeSG treatment induces immunogenic cell death (ICD). MeSG induced significant cell surface calreticulin (CRT) exposure in a time and concentration dependent manner as well as increased phagocytosis of tumor cells by dendritic cells in vitro. Interestingly, this CRT exposure differs from the canonical pathway in several aspects. MeSG does not cause ER stress and does not require PERK to induce CRT exposure. Caspase inhibitors partially rescue cells from MeSG-induced apoptosis, but fail to reduce CRT exposure. MeSG does not cause ERp57 exposure and the absence of ERp57 expression does not reduce CRT exposure. Finally, an intact ER to Golgi transport system is required for this phenomenon. These results lend support to the development of the schweinfurthin family as drugs to enhance clinical response to immunotherapy and highlight the need for additional research on the mechanisms of ICD induction.

Emily J. Koubek, Jillian S. Weissenrieder, Luz E. Ortiz, Nnenna Nwogu, Alexander M. Pham, J. Dylan Weissenkampen, Jessie L. Reed, Jeffrey D. Neighbors, Raymond J. Hohl, Hyun Jin Kwun

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin cancer predominantly caused by the human Merkel cell polyomavirus (MCPyV). Treatment for MCC includes excision and radiotherapy of local disease, and chemotherapy or immunotherapy for metastatic disease. The schweinfurthin family of natural compounds previously displayed potent and selective growth inhibitory activity against the NCI-60 panel of human-derived cancer cell lines. Here, we investigated the impact of schweinfurthin on human MCC cell lines. Treatment with the schweinfurthin analog, 5′-methylschweinfurth G (MeSG also known as TTI-3114), impaired metabolic activity through induction of an apoptotic pathway. MeSG also selectively inhibited PI3K/AKT and MAPK/ERK pathways in the MCPyV-positive MCC cell line, MS-1. Interestingly, expression of the MCPyV small T (sT) oncogene selectively sensitizes mouse embryonic fibroblasts to MeSG. These results suggest that the schweinfurthin family of compounds display promising potential as a novel therapeutic option for virus-induced MCCs.

Jillian S. Weissenrieder, Jessie L. Reed, George Lucian Moldovan, Martin T. Johnson, Mohamed Trebak, Jeffrey D. Neighbors, Richard B. Mailman, Raymond J. Hohl

Dopamine D₂-like receptor antagonists have been suggested as being potential anticancer therapeutics with specific utility for central nervous system cancers due to their ability to cross the blood-brain barrier. Despite a plethora of data reporting anticancer effects for D₂R antagonists in cell or animal studies, the ligand concentrations or doses required to achieve such effects greatly exceed the levels known to cause high degrees of occupancy of the D₂ receptor. To resolve this conundrum, we interrogated a panel of glioblastoma multiforme (GBM) cell lines using D₂ antagonists of varying chemotype. We studied the cytotoxic effects of these compounds, and also ascertained the expression of D₂ receptors (D₂R) on these cells. Although several chemotypes of D₂R antagonists, including phenothiazines and phenylbutylpiperidines, were effective against GBM cell line cultures, the highly selective antagonist remoxipride had no anticancer activity at biologically relevant concentrations. Moreover the D₂R antagonist-induced cytotoxicity in monolayer cultures was independent of whether the cells expressed D₂R. Instead, cytotoxicity was associated with a rapid, high-magnitude calcium flux into the cytoplasm and mitochondria, which then induced depolarization and apoptosis. Blocking this flux protected the GBM cell lines U87MG, U251MG, and A172. Together, these data suggest that the cytotoxicity of these D₂R antagonists involves calcium signaling mechanisms, not D₂R antagonism. Repurposing of existing drugs should focus on the former, not latter, mechanism.

Jillian S. Weissenrieder, Jessie L. Reed, Michelle V. Green, George Lucian Moldovan, Emily J. Koubek, Jeffrey D. Neighbors, Raymond J. Hohl

Background: Glioblastoma multiforme (GBM) is a common and lethal cancer of the central nervous system. This cancer is difficult to treat because most anticancer therapeutics do not readily penetrate into the brain due to the tight control at the cerebrovascular barrier. Numerous studies have suggested that dopamine D₂ receptor (D₂R) antagonists, such as first generation antipsychotics, may have anticancer efficacy in vivo and in vitro. The role of the D₂R itself in the anticancer effects is unclear, but there is evidence suggesting that D₂R activation promotes stem-like and spheroid forming behaviors in GBM. Objectives: We aimed to observe the role of the dopamine D₂R and its modulators (at selective concentrations) in spheroid formation and stemness of GBM cell line, U87MG, to clarify the validity of the D₂R as a therapeutic target for cancer therapy. Results: D₂R antagonists thioridazine, pimozide, haloperidol, and remoxipride decrease spheroid formation behaviors at a selective 100 nmol/L concentration, while D₂R agonists PHNO, sumanirole, and ropinirole increase the formation of spheroids. Similarly, 100 nmol/L remoxipride decreased sphere-forming cell frequency. These results were recapitulated with genetic overexpression and knockdown of the D₂R, and combination experiments indicate that the D₂R is required for the effects of the pharmacological modulators. Furthermore, spheroid proliferation and invasive capacity increased under treatment with 100 nmol/L sumanirole and decreased under treatment with 100 nmol/L thioridazine. Expression levels of the stemness markers Nestin and Sox2, as well as those of differentiation marker glial fibrillary acidic protein, were not altered by 100 nmol/L thioridazine or sumanirole for 72 h or continuous treatment with these compounds for 7 days during a spheroid formation assay. Conclusions: Signaling activity of the dopamine D₂R may be involved in the spheroid formation phenotype in the context of the U87MG cell line. However, this modulation may not be due to alterations in stemness marker expression, but due to other factors that may contribute to spheroid formation, such as cell-cell adhesion or EGFR signaling.

Kathleen M. Kokolus, Jeremy S. Haley, Emily J. Koubek, Raghavendra Gowda, Saketh S. Dinavahi, Arati Sharma, David F. Claxton, Klaus F. Helm, Joseph J. Drabick, Gavin P. Robertson, Jeffrey D. Neighbors, Raymond J. Hohl, Todd D. Schell

Jillian S. Weissenrieder, Jacqueline E. Reilly, Jeffrey D. Neighbors, Raymond J. Hohl

Emily J. Koubek, Jillian S. Weissenrieder, Jeffrey D. Neighbors, Raymond J. Hohl

The schweinfurthin family of compounds displays exciting potent and differential cytotoxicity against human cancer cell lines. Currently, the effect of schweinfurthins on tumor development and progression is being explored in animal models of cancer with promising results. The first schweinfurthin family member, vedelianin, was isolated in 1992, followed by other schweinfurthins in 1998. This opened up the door for the synthesis of additional analogs. At present, the focus of research lies on delineating the mechanism of schweinfurthin action and identifying the nature of sensitivity. It appears that many of the intracellular effects of schweinfurthins are due to, or impacted by, the effect of schweinfurthins on lipid metabolism, synthesis, and homeostasis. These effects include impaired trafficking from the trans-golgi network, disruption of lipid rafts, changes in oxysterol-binding protein activity, and interference with the isoprenoid biosynthesis pathway (IBP). Cancer cells are known to rely heavily on fatty acid, lipid, and sterol synthesis for growth and proliferation. Therefore, compounds that target these needs, such as schweinfurthins, display promise as novel therapeutics. This timely review will take an in-depth look at the history of schweinfurthins, their synthesis, where the research presently stands, and the questions that remain.