Analysis of acquired resistance to metronomic oral topotecan chemotherapy plus pazopanib after prolonged preclinical potent responsiveness in advanced ovarian cancer

Abstract

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy—the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. the anti- VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.

In Vivo Assessment of Ovarian Tumor Response to Tyrosine Kinase Inhibitor Pazopanib by Using Hyperpolarized13C-Pyruvate MR Spectroscopy and18F-FDG PET/CT Imaging in a Mouse Model

“…In summary, our findings suggest that hyperpolarized 13 C-pyruvate-tolactate conversion can serve as an early indicator of response to multityrosine kinase inhibitor pazopanib in an ovarian cancer model and that evaluation of late events in glycolysis, such as by hyperpolarized 13 C-pyruvate MR, may be predictive of response even when the Pazopanib can affect vascular endothelial growth factor receptors on ovarian tumor cells and inhibit SKOV3 cell proliferation (29) but the effects in tumors are primarily paracrine and effect angiogenesis (30). Pazopanib inhibits ovarian tumor growth through its antiangiogenic activity and this may have led to an altered hypoxic state.…”

Potent efficacy of metronomic topotecan and pazopanib combination therapy in preclinical models of primary or late stage metastatic triple-negative breast cancer

“…In vitro assessment of cell proliferation inhibition by topotecan, pazopanib and their combination on triple negative breast cancer cell line MDA-MB 231 cell line [ 63 , 64 ] and its highly metastatic variant called MDA-MB 231/LM2-4 [ 40 ] and on endothelial cells for 72h [ 65 ] and 144h [ 58 ] was performed by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS; Promega) [ 66 ]; apoptosis measurements were made by Cell Death Detection ELISA Plus kit (Roche Diagnostics, Laval, QC, Canada) and bromodeoxyuridine (BrdU) incorporation analysis; HIF1α and ABCG2 gene expression was obtained with Real-time PCR analysis and their protein levels evaluation was performed by Western Blot assay; intracellular accumulation of topotecan was measured by HPLC analysis as previously described [ 67 , 68 ]. The details of these experiments are reported in the Supplementary Materials and Methods .…”

Resistance to metronomic chemotherapy and ways to overcome it

“…Cruz-Muñoz et al studied SKOV-3-13 human ovarian cancer cells made resistant to metronomic topotecan plus the small molecule VEGFi pazopanib in vivo [51]. Oligo-microarray analysis of these variants revealed a number of upregulated genes implicated in resistance to various chemotherapeutic agents, namely alpha B crystallin (CRYAB), heat shock 27 kDa protein 2 (HSPB2), transketolase-like 1 (TKTL1), and the cytochrome P450 member CYP1B1.…”

“…To name a few, MC was successfully combined with standard anticancer treatment modalities such as VEGFi [13,51], conventional MTD chemotherapy [65], targeted therapies [33,66], and radiation [67]. The immunomodulatory activities of MC were exploited to enhance different types of cancer immunotherapy [68].…”