Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma

Abstract

Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug Resistant Human Breast Cancer

“…It is clear that the inherent level of APC1 phosphorylation in MCF7 chemoresistant cells is similar to that in unselected parental cells treated with APCIN, and that the level in resistant cells normalizes up to parental levels when treated with M2I-1. This is consistent with our findings that M2I-1 exposure reduced APC substrate protein levels, a marker of APC activation, in canine OSW lymphoma cells selected for resistance to DOX [9]. This also suggests that the APC defect is fully reversible, which is clinically important when considering this as a potential treatment target.…”

“…Our recent work supports observations that the APC is inhibited in aggressive cancer cells and that APC activation can reverse drug resistance [9]. We demonstrated that metformin treatment, when combined with CHOP chemotherapy (Cyclphosphamide, Doxorubicin (DOX), Vincristine, and Prednisone), reversed MDR lymphoma in canines in vivo, as it did in vitro [27]; all dogs tested showed reduced expression of markers of MDR and one canine went into remission [9]. We found that tumor samples derived from the canines expressed high levels of all 33 different APC substrate mRNAs that were present on the canine microarray, and that metformin treatment reduced all levels to normal, indicating that metformin induced APC activity.…”

“…Our recent work supports observations that the APC is inhibited in aggressive cancer cells and that APC activation can reverse drug resistance [9]. We demonstrated that metformin treatment, when combined with CHOP chemotherapy (Cyclphosphamide, Doxorubicin (DOX), Vincristine, and Prednisone), reversed MDR lymphoma in canines in vivo, as it did in vitro [27]; all dogs tested showed reduced expression of markers of MDR and one canine went into remission [9].…”

“…The reciprocal co-immunoprecipitation experiment was performed (Figure 1F) with consistent results, again showing decreased CDC27 associations with CDC20 specifically in resistant cells. Furthermore, we used antibodies against protein markers of MDR (BCRP, MDR-1 and TFPI) [9,27,32,38] and DNA damage (gH2AX) [39] to confirm that the TAM selected cells are indeed multiple drug resistant and experiencing higher levels of DNA damage (Figure 2A,B; two separate experiments done independently are shown in A and B). Our data shown here indicates that the reduction of CDC20 and CDH1 interactions with CDC27 are not cell cycle specific and reflect an impairment of both APC CDC20 and APC CDH1 .…”

“…An additional mechanism driving MDR that has come to light in recent years is impairment of Anaphase Promoting Complex (APC) activity that is associated with drug resistant cancer [9,10]. Numerous studies have observed that APC inhibition is linked with aggressive cancer development in vitro and in vivo [11][12][13][14][15].…”