In vitro study to assess the efficacy of CDK4/6 inhibitor Palbociclib (PD‐0332991) for treating canine mammary tumours

Abstract

Therapy of canine mammary tumours (CMTs) with classical antitumour drugs is problematic, so better therapeutic options are needed. Palbociclib (PD-0332991) is an innovative and effective anticancer drug for the treatment of breast cancer in women. Palbociclib is an inhibitor of cyclin-dependent kinase 4 (CDK4) and CDK6, which are key regulators of the cell cycle machinery and thus cell proliferation. In the present in vitro study, we investigated whether Palbociclib also represents a candidate drug to combat CMT. For this purpose, the effect of Palbociclib was analysed in P114 and CF41 cells, two CMT cell lines with an endogenous CDK4/6 co-expression. Incubation of P114 and CF41 cells with Palbociclib resulted in a dose-and timedependent loss of phosphorylated retinoblastoma protein (pRb), a classical CDK4/6 substrate within the cell cycle machinery. Moreover, treatment of CMT cells with Palbociclib-induced cell cycle arrest affected cell viability, prevented colony formation and impaired cell migration activity. Palbociclib also inhibited the growth of P114 and CF41 cell spheroids. Immunohistochemical analysis of canine patient samples revealed a consistent expression of CDK6 in different canine mammary carcinoma types, but an individual and tumour-specific expression pattern of phosphorylated pRb independent of the tumour grade. Together, our findings let us suggest that Palbociclib has antitumour effects on CMT cells and that canine patients may represent potential candidates for treatment with this CDK4/6 inhibitor. K E Y W O R D S canine mammary tumour, CDK4/6, cell cycle, migration, Palbociclib

mRNA sequencing analysis and growth inhibitory effects of palbociclib on cell lines from canine histiocytic proliferative disorders

“…Rb is phosphorylated by CDK4/6 at four sites (S780, S795 and S807/811), and palbociclib, a CDK4/6 inhibitor, has been shown to inhibit the phosphorylation of these sites 42 . The efficacy of palbociclib in the treatment of human breast carcinoma, lung carcinoma and squamous cell carcinoma and canine mammary tumours has been validated 25,42,43 . In the present study, palbociclib was selected as a therapeutic drug candidate for canine histiocytic proliferative disorders, and its growth inhibitory effects were verified in vitro and in xenograft mouse models.…”

“…42 The efficacy of palbociclib in the treatment of human breast carcinoma, lung carcinoma and squamous cell carcinoma and canine mammary tumours has been validated. 25,42,43 In the present study, palbociclib was selected as a therapeutic drug candidate for canine histiocytic proliferative disorders, and its growth inhibitory effects were verified in vitro and in xenograft mouse models. Since the average IC 50 value of palbociclib was higher in the disseminated HS/SyH group (15.5 μM) than in the localized HS group (10.5 μM) and LCH cell line (7.6 μM), the growth inhibitory effects of palbociclib in xenograft mouse models was verified using the disseminated HS cell line (FCR-DHS04).…”

“…Based on the results of IPA and FPKM values, immunohistochemistry (IHC) using a mouse anti-Rb antibody (1:200; clone 4H1, Cell Signaling Technology, Tokyo, Japan) 25,26 and rabbit anti-phosphorylated Rb (pRb, Ser807/811) antibody (1:200; clone D20B12, Cell Signaling Technology) 27 was performed. The mouse xenograft tissues of 6 cell lines produced in previous studies 15,19,20 and tissues of 38 canine histiocytic proliferative disorders cases (Table S2) were examined.…”

H2AFZ: A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment

“…Specifically, the anti-proliferative effects of CDK4/6 inhibitors on both tumor cells and Tregs appear to be associated with reduced activity of DNA methyltransferase 1, that is encoded by the E2F target gene DNMT1 (29). In dogs, CDK4/6 inhibitors have been recently proposed for the treatment of canine mammary tumors, based on promising in vitro investigations (30).…”

Decreased sensitivity of cyclin-dependent kinase 4/6 inhibitors, palbociclib and abemaciclib to canine lymphoma cells with high p16 protein expression and low retinoblastoma protein phosphorylation

“…In human cancer cell lines, it has been reported that CDK4/6i can effectively inhibit the proliferation of tumor cells that lose endogenous inhibition of CDK4/6 due to p16 methylation or deletion [ 15 , 16 ]. In canine studies, CDK4/6i and palbociclib have antitumor effects on canine mammary tumor cells [ 25 ] and could potentially be used as new anti-cancer treatments for canine melanoma [ 3 ]; however, there is no data available simultaneously analyzing the expression of p16 protein and phosphorylated pRb (pRb-P).…”