Protein Ligands in the Secretome of CD36+ Fibroblasts Induce Growth Suppression in a Subset of Breast Cancer Cell Lines

Abstract

Reprogramming the tumor stroma is an emerging approach to circumventing the challenges of conventional cancer therapies. This strategy, however, is hampered by the lack of a specific molecular target. We previously reported that stromal fibroblasts (FBs) with high expression of CD36 could be utilized for this purpose. These studies are now expanded to identify the secreted factors responsible for tumor suppression. Methodologies included 3D colonies, fluorescent microscopy coupled with quantitative techniques, proteomics profiling, and bioinformatics analysis. The results indicated that the conditioned medium (CM) of the CD36+ FBs caused growth suppression via apoptosis in the triple-negative cell lines of MDA-MB-231, BT549, and Hs578T, but not in the ERBB2+ SKBR3. Following the bioinformatic analysis of the CM of CD36+ versus CD36− FBs, we determined KLF10 as one of the transcription factors responsible for growth suppression. We also identified FBLN1, SLIT3, and PENK as active ligands, where their minimum effective concentrations were determined. Finally, in MDA-MB-231, we showed that a mixture of FBLN1, SLIT3, and PENK could induce an amount of growth suppression similar to the CM of CD36+ FBs. In conclusion, our findings suggest that these ligands, secreted by CD36+ FBs, can be targeted for breast cancer treatment.

CD36+ Fibroblasts Secrete Protein Ligands That Growth-Suppress Triple-Negative Breast Cancer Cells While Elevating Adipogenic Markers for a Model of Cancer-Associated Fibroblast

“…Hence, we have sought to trigger tumor suppression by restoring the normal epithelia–FB communications. Here, we have extended our previous findings that FBs expressing CD36, a receptor initially discovered through its roles in lipid uptake and adipogenesis, secrete a cocktail of protein ligands that inhibit the growth of certain types of breast cancer [ 9 , 10 ]. CD36 is expressed in FBs of normal mammary glands [ 9 ], where normal mammary FBs are known to exert anti-tumor functions through paracrine signaling [ 11 , 12 ].…”

“…Here, we have extended our previous findings that FBs expressing CD36, a receptor initially discovered through its roles in lipid uptake and adipogenesis, secrete a cocktail of protein ligands that inhibit the growth of certain types of breast cancer [ 9 , 10 ]. CD36 is expressed in FBs of normal mammary glands [ 9 ], where normal mammary FBs are known to exert anti-tumor functions through paracrine signaling [ 11 , 12 ]. Downregulation of CD36 in FBs is one of the CAF markers [ 5 ], and in the normal mammary gland, it is often associated with high mammographic density (MD) [ 13 ].…”

“…Earlier, we showed that the coculture of the triple-negative breast cancer (TNBC) MDA-MB-231 or Luminal A MCF7 with CD36 + fibroblasts (FBs) induced growth suppression in TNBC MDA-MB-231 and reversion of basal and lateral polarity in the Luminal A MCF7 [ 10 ]. Subsequently, we also showed that the conditioned medium (CM) of the CD36 + fibroblasts (FBs) also induces growth suppression in MDA-MB-231 [ 9 ]. Because the CM can be concentrated and used as a positive control for future comparative studies, it is important to examine the impact of its concentration on colony formation for a non-transformed cell line such as MCF10A, which is a mammary epithelial cell line.…”

“…Regardless, the sensitivity of these cell lines to RPs did not appear to be cell-line specific, and in all cases, the cocktail of ligands showed higher growth suppression, which suggests that these recombinant proteins have an additive growth suppression effect. The control for this experiment was non-malignant MCF10A which showed an approximately 20% reduction in the rate of colony formation as per our previous manuscript ( Supplementary Results ) [ 9 ].…”

“…Next, we performed comparative proteomic profiling of the secretome of CD36 + vs. CD36 − FBs and identified a number of candidate protein ligands. After the functional screening, we narrowed down active ligands to SLIT3, PENK, and FBLN-1, and determined their minimum effective concentrations [ 9 ]. In the present study, we show that each of the three ligands could also reprogram CAFs and induce their transdifferentiation by overexpressing CD36 and FABP4 while strongly suppressing the growth of breast cancer cells.…”