Vitamin D analogs in combination with palbociclib as treatment for triple-negative breast cancer – preclinical evaluation and pathway analysis

1,25(OH)2D3, the biological active form of vitamin D3, is an important regulator of calcium and phosphate homeostasis in the body. By binding to the vitamin D receptor (VDR) and subsequent heterodimerization with the retinoid X receptor (RXR), this complex will migrate to the nucleus and bind to vitamin D responsive elements (VDREs) in regulatory regions of target genes to regulate gene transcription. Next to calcium and phosphate homeostasis, different biological processes such as cell proliferation, differentiation, immune modulation, apoptosis and cell metabolism are regulated by 1,25(OH)2D3. These effects are induced in normal as well as in malignant cells including breast cancer (BC) cells. To induce these antiproliferative effects, supraphysiological doses of 1,25(OH)2D3 are needed which cause calcemic side effects when applied in vivo. To overcome this problem, vitamin D3 analogs were generated, which have a better dissociation between their antiproliferative effect and calcemic activity. One such potent analog synthesized in our laboratory in collaboration with the Department of Organic Chemistry of Ghent University is inecalcitol. This analog has an increased antiproliferative capacity and reduced calcemic activity compared to natural 1,25(OH)2D3. Inecalcitol is able to reduce BC cell proliferation by blocking the G1-S phase transition of the cell cycle and thereby reducing in vivo tumor growth in a cell line-derived xenograft model of estrogen receptor (ER)-positive BC.

BC is a highly prevalent disease with 2.3 million diagnoses each year worldwide, so there is a high need for the development of novel therapies. As dysregulation of cell cycle genes is often present in BC, selective cell cycle inhibitors such as palbociclib (CDK4/6 inhibitor) have been generated and are used in the clinic for the treatment of hormonal receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative metastatic BC in combination with hormonal therapy. Palbociclib is currently not implemented for the treatment of triple-negative breast cancer (TNBC).

Because the VDR is expressed in approximately 60 % of invasive breast carcinomas, we hypothesized that targeting the vitamin D signaling pathway might be an effective addition to treatment regimens in BC patients. Therefore, we investigated if the combination of inecalcitol and palbociclib treatment potentiated the antiproliferative effect of both single compounds and studied if the addition of inecalcitol increased the sensitivity of TNBC to palbociclib treatment. Furthermore, we elucidated the pathways affected by the combination therapy in HR+ and TNBC cells by performing tracer metabolomics.

In the first aim of this study, we investigated the effect of inecalcitol and palbociclib mono and combination treatment in different BC subtypes: ER+ BC (T47D-MCF7) and TNBC (BT20, HCC1143 and Hs578T). By using different assays, we observed that combining inecalcitol with palbociclib reduced cell proliferation and growth mainly in ER+ BC cells and to a lesser extent in TNBC. Inecalcitol alone and combined with palbociclib also reduced cell survival in ER+ MCF7 cells, while no effect was observed in the TNBC BT20 cell line. This increased antiproliferative effect was confirmed by cell cycle analysis with PI FACS staining and by western blot detection of cell cycle regulators (p)-Rb and (p)-CDK2. The combination therapy blocked the G1 to S phase transition of the cell cycle by downregulation of (p)-Rb and (p)-CDK2. A more detailed analysis of the Akt/PI3K/mTOR cell survival pathway showed that this pathway was regulated differently in ER+ MCF7 than in TNBC BT20 cells. Indeed, the combination therapy increased the levels of (p)-mTOR and (p)-4E-BP1 in MCF7 cells while in BT20 cells, these levels were downregulated. Still, in both cell lines, treatment with the combination therapy upregulated mitochondrial ROS levels and increased mitochondrial content.

In the second aim of this study, we investigated the effect of the combination therapy in vivo using a cell line-derived xenograft mouse model. We generated models derived from either ER+ MCF7 or TNBC BT20 cells. Dose titration studies were performed in order to define the optimal combination dose without inducing calcemic side effects. Again, treatment with the inecalcitol and palbociclib combination therapy reduced tumor volume and weight in an MCF7 cell line-derived xenograft model, while there was no effect in the TNBC xenograft model. This confirms the findings observed in the in vitro analysis as also the cell cycle regulatory proteins p-Rb and p-CDK2 were downregulated in the MCF7 xenograft tumors. The decreased cell proliferation capacity of the MCF7 xenografts was confirmed by a reduced level of Ki67-positive cells in the combination-treated xenografts. Although, we did not observe any calcemic side effects during the dose titrations studies, treatment with inecalcitol and even more with the combination therapy increased serum calcium and urine levels in both xenograft models. Yet, there were no changes in bone volume and femur calcium content observed after either treatment regimen, suggesting that there was no calcium resorption from the bone.

Finally, in the third aim, we further elaborated the effect of the mono and combination treatment on cell metabolism more specifically on the effect of glutamine metabolism in BC cells. By labeling the cells with [U-13C]-glutamine, we analyzed the changes induced by the treatment into the tricarboxylic acid (TCA) cycle, amino acids and nucleotide synthesis. By analyzing two different time points, we could differentiate between direct and indirect effects. We could not observe any major alterations after 24 h of treatment, while after 72 h of treatment glutamine incorporation tended to be reduced in the oxidative TCA cycle in MCF7 cells. In addition, palbociclib treatment reduced glutamine incorporation into the reductive TCA cycle, whereas this effect was not observed in BT20 cells. The combination therapy significantly reduced the labeling of glutamine into the pyrimidine nucleotides UTP and CTP, also in BT20 cells. Interestingly, the combination therapy reduced the levels of aspartate in MCF7 cells, while in BT20 cells, these levels were upregulated by inecalcitol. These changes in cell metabolism also correlated with the difference in responsiveness between the two cell lines and suggest that these effects are mediated indirectly by cell cycle inhibition.

In conclusion, this study demonstrated the antitumor activity of combining a vitamin D3 analog with a CDK4/6 inhibitor in ER+ BC cells. Furthermore, novel insights in the underlying signaling mechanism of this treatment strategy were provided in different BC subtypes.