In this current study we not
In this current study, we not only demonstrated the growth-inhibitory effects of the 2nd generation analog SL-1-39 on the HER2+ breast cancer cells, MDA-MB-453 and SKBR3 (Table 1), but we also demonstrated that the decrease in cell number was associated with an interruption of brilliant blue progression through the down-regulation of multiple key cell cycle regulators, such as cyclin A and D1. These observations are consistent with previous studies carried out on the parental drug, SHetA2, and another 2nd generation analog, SL-1-18, both of which have been shown to decrease cyclin D1 expression and subsequently arrest cells at the G1 phase [24,36,37]. We speculate that the post-transcriptional decrease in cyclin D1 levels (see Fig. 2) is likely attributed to reduction in MAPK activity mediated by SL-1-39 treatment. Alternatively, as seen with SL-1-18, SL-1-39 may increase the degradation of the cyclin D1 protein via the proteasome and/or lysosome, although this was not observed in any of the MG132 or BAF experiments (data not shown).
The most notable results presented in this study demonstrate that SL-1-39 reduces protein expression of HER2 by inducing lysosomal degradation (Fig. 4, Fig. 5, Fig. 6). In the HER2+ breast cancer cell lines MDA-MB-453, SKBR3, T47D, and BT-474, SL-1-39 decreased HER2 protein expression and the HER2-associated phosphorylation of MAPK in a time-dependent manner (Fig. 4, Fig. 5). Though HER2 protein levels decreased within 2 h of exposure (Fig. 5), HER2 mRNA levels remained unchanged, suggesting that the decrease in HER2 is associated with degradation of the HER2 protein. Curiously, we found that cells with lower levels of HER2 (e.g. T47D) were more sensitive to SL-1-39-induced HER2 degradation than cells with high levels of HER2 (e.g. SKBR3).
This is the first study to show that a flexible heteroarotinoid analog can target the HER2 signaling pathway, and our results suggest that this decrease is associated with the lysosomal degradation pathway. Such a finding is not completely without precedence, as other anti-HER2 agents, including trastuzumab, are known to bind selectively to the extracellular domain of HER2 and promote HER2 degradation by receptor-mediated endocytosis into the lysosome [29,42,43]. How SL-1-39 triggers the lysosomal degradation pathway remains unclear. However, we speculate that since the normal turnover of most plasma membrane receptors requires lysosomal targeting via the ubiquitination pathway , and HER2 has been shown in previous studies to undergo ubiquitin-dependent lysosomal degradation [, , ], SL-1-39 may initiate degradation of HER2 by triggering its ubiquitination. This supposition is further supported by our recently published study on the sister compound, SL-1-18, which promotes the ubiquitination and subsequent degradation of the cytoplasmic receptor ERα.
The observation that SL-1-39 down-regulates HER2 faster than trastuzumab (2 vs. 24 h, respectively) [, , , ] is quite promising. Especially intriguing is the idea that SL-1-39 might be an effective therapeutic against certain trastuzumab-resistant breast cancers, particularly those that are associated with a truncated HER2 (p95-HER2) or ERBB2 mutations . Since SL-1-39 works intracellularly, the lack of an extracellular domain in p95-HER2 should have little to no effect on its activity. Similarly, the most common ERBB2 mutations in breast cancer patients do not affect the HER2 ubiquitination docking site, tyrosine-1112, which allows the polyubiquitination of HER2 via the E3 ubiquitin ligase (i.e. c-Cbl) . In short, our encouraging findings warrant further development of SL-1-39 as a potential therapeutic for HER2+ breast cancer.
Conflicts of interest
Acknowldegements This work was supported by grant funding from Dominican University, School of Health and Natural Sciences Research grant to M.C.L. and Touro University of California, College of Pharmacy, Intramural Research Award Program (IRAP) to S.L.