Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment
Authors: Bowers Laura W. , Gung Joseph , Lineberger Claire G. , Hursting Stephen D. 
Published: August 2019

Epidemiological studies have linked obesity to a greater risk of breast cancer recurrence and shorter disease-free survival, across breast cancer subtypes. This includes triple-negative breast cancer (TNBC), which has few treatment options beyond the traditional cytotoxic chemotherapies. Unfortunately, numerous studies have found that obese patients do not respond as well as normal weight women to chemotherapy, including taxane, anthracycline, and platinum-based anti-neoplastic drugs. These findings suggest that obesity may promote resistance to multiple types of chemotherapy via a common mechanism.

One factor that may promote chemotherapy resistance is an enrichment in the tumor-initiating cell (TIC) population. The TIC model posits that these cells, an intra-tumoral subpopulation with stem cell-like properties, are the primary drivers of tumor initiation, growth, metastasis, and chemotherapy resistance. In breast cancer patients, this hypothesis has been supported by associations between TIC enrichment and poor patient prognosis. Our laboratory has previously demonstrated that obesity increases the expression of TIC-related genes in pre-clinical transplant models of TNBC. This was accompanied by an upregulation in markers of epithelial-to-mesenchymal transition (EMT), a key developmental program that is closely linked with TIC enrichment and metastasis. Others have reported a link between leptin signaling and TIC enrichment, and elevated systemic leptin levels as well as increased tumor leptin and leptin receptor expression levels are associated with a worse breast cancer prognosis. Consequently, it is highly probable that leptin signaling is a key contributor to obesity-associated chemotherapy resistance in TNBC via promotion of TIC enrichment.

In the current study, we first utilized MMTV-Wnt-1 transgenic mice, a model of TNBC, and in vitro models of TNBC to test the hypothesis that increased leptin signaling drives obesity-associated TNBC development via promotion of TIC enrichment and EMT. We then employed an orthotopic transplant model of TNBC, derived from the MMTV-Wnt-1 mouse, to examine the role of leptin-induced TIC enrichment in obesity-associated resistance to the chemotherapy drug docetaxel. Specifically, female MMTV-Wnt-1 mice were randomized to a control (10% kcal from fat) or a diet-induced obesity (DIO, 60% kcal from fat) regimen (n=15/diet group), both fed ad libitem. Mice were followed for up to 42 weeks and euthanized when tumor diameter was &gt;1.5 cm or at study termination, whichever occurred first. Total RNA was isolated from tumor samples using TRIzol Reagent, RNA quality assessed by Agilent 2100 Bioanalyzer, and randomly selected samples from control (n=5) and DIO (n=6) mice prepared for sequencing using the Illumina TruSeq RNA Library Preparation Kit. RNA libraries were then sequenced on the Illumina HiSeq 2000 instrument. RNA sequencing reads were aligned to Mouse Genome Ensembl GRCm38 using TopHat (version 2.1.0), the differential levels of transcripts quantified by Cuffdiff/Cufflink 2.2.1, and normalized FPKM were generated by Cufflinks as representative of gene expression level. Genes were entered into Qiagen’s Ingenuity Pathway Analysis (IPA). The expression level of genes of interest were validated by quantitative RT-PCR. A colorimetric ALDH Activity Assay Kit was used to assess tumor ALDH activity.

Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice (E-Wnt and M-Wnt), and human MDA-MB-231 TNBC cells were used for in vitro studies. The impact of leptin treatment (100, 200, and 400 ng/ml) on mammosphere formation in these 3 cell lines was assessed. Viability, migration, invasion, and TIC/EMT-related genes expression in the 3 cell lines was also measured following exposure to a 2% concentration (in serum-free media) of serum collected from wild-type C57BL/6 mice maintained on the DIO or control diet regimens for 14 weeks. E-Wnt cells were stably transfected with Lepr shRNA (EWnt-L1 and EWnt-L2) or scrambled shRNA (EWnt-S) plasmids and mammosphere formation, viability, migration, invasion, and TIC/EMT-related gene expression was assessed. Finally, wild-type C57BL/6 mice were randomized to the control or DIO regimens, maintained on these diets for 15 weeks, then orthoptically injected with EWnt-L1 cells in the 4th mammary fat pad (MFP) and EWnt-S cells in the 9th MFP (1 x 10^6 cells for both). Mice in each diet group were further randomized to vehicle or docetaxel (20 mg/kg IV, once weekly for 3 weeks) treatments. Tumors were palpated twice/week, and vehicle or drug treatment initiated when the larger of the 2 tumors measured ~200 mm3. Each mouse was euthanized when at least 1 of the 2 tumors measured 1.5 cm in diameter. Total RNA was isolated from the tumors and assessed for quality as described above, and the transcriptome of both tumors from randomly selected mice (n=6/group) was analyzed by Applied Biosystems’ Mouse Clariom S Assay HT. Results were entered into ThermoFisher Scientific’s Transcriptome Analysis Console (TAC) software.

MMTV-Wnt-1 mice fed a DIO versus control diet regimen had significantly greater body weight and percent body fat at final assessment (P&lt;0.01 for both). DIO mice also had significantly reduced tumor-free survival compared with controls (P&lt;0.05). Gross necropsy detected no liver or lung metastases in either diet group. IPA analysis of RNA sequencing data indicated significant upregulation of protumorigenic pathways characterized as ‘regulation of EMT’ (P=0.003) and ‘human embryonic stem cell pluripotency’ (P=0.026) and identified Lep as a highly significant (P&lt;0.001) upstream master regulator. Validation of TIC/EMT-related gene expression via quantitative RT-PCR indicated that tumors from DIO mice versus controls displayed significantly upregulated Aldh1a1, Pou5f1 (both P&lt;0.01), Akt3, Pik3r1, Twist1, Twist2, Foxc2, and Vim (each P&lt;0.05) as well as downregulated Cdh1 (P&lt;0.05). Tumor ALDH activity (a measure of TIC enrichment) and Lepr expression were also greater in DIO mice than controls (P&lt;0.05).

In vitro, leptin treatment significantly increased mammosphere formation in E-Wnt, M-Wnt, and MDA-MB-231 cells in comparison to vehicle (P&lt;0.05), while the Lepr knockdown EWnt-L1 and EWnt-L2 cells formed significantly fewer mammospheres in comparison to EWnt-S cells (P&lt;0.001). Exposure to DIO mouse serum significantly increased viability, migration, invasion, and the expression of several TIC/EMT-related genes in all 3 cells lines, relative to control mouse serum (P&lt;0.05). Lepr knockdown in the E-Wnt cells decreased the effects of DIO mouse serum on cell viability, migration, invasion, and a subset of TIC/EMT related genes (P&lt;0.05), such that there was no longer a significant difference between the DIO and control conditions for cell viability, migration, and expression of Akt3, Foxc2, Twist2, and Vim.

The wild-type C57BL/6 mice fed a DIO versus control diet regimen had significantly greater body weight and percent body fat at euthanization (P&lt;0.01 for both), with no differences between the vehicle and docetaxel mice within each diet group. Docetaxel treatment in the DIO mice significantly reduced the tumor growth rate in the EWnt-L1 tumors (P&lt;0.05), but not the EWnt-S tumors, in comparison to vehicle treatment. Tumor growth rate was defined as the difference between final ex vivo tumor volume and estimated tumor volume at the start of treatment, divided by days elapsed between treatment initiation and euthanization. In contrast, docetaxel treatment significantly reduced the growth rate of both EWnt-L1 and EWnt-S tumors in the control diet-fed mice (P&lt;0.05 for both). Analysis of tumor microarray data via TAC indicated that the DIO regimen, relative to control, promoted a significant upregulation in EWnt-S tumor expression of genes in the ‘PluriNetWork’ (29 genes, P&lt;0.05) and ‘ESC Pluripotency’ (18 genes, P&lt;0.001) pathways. Intriguingly, this modulation of TIC-related genes was seen in DIO mice that received docetaxel, but not vehicle treatment. In addition, the DIO regimen did not significantly affect these pathways in the EWnt-L1 tumors from both vehicle and docetaxel-treated mice. Finally, we observed that the EWnt-L1 tumors, in comparison to EWnt-S tumors, in DIO mice demonstrated significant downregulation in ‘PluriNetWork’ pathway gene expression under both vehicle (59 genes, P&lt;0.05) and docetaxel (77 genes, P&lt;0.05) treatment conditions. No significant differences between EWnt-L1 and EWnt-S cells in TIC-related gene expression were observed in the control mice.

These results suggest that obesity and docetaxel treatment together promote TIC enrichment in E-Wnt tumors via a leptin-mediated mechanism, which results in resistance to docetaxel. Our findings will inform the design of future mechanistic and translational studies that further delineate the pathway(s) by which obesity-induced TIC enrichment promotes chemotherapy resistance, with the ultimate goal of developing a more efficacious pharmaceutical treatment regimen for the obese TNBC patient population and identifying biomarkers for the detection of patients at highest risk of poor outcome.

Citation Format: Laura W. Bowers, Joseph Gung, Claire G. Lineberger, Stephen D. Hursting. Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY28-04.