Therapeutic Potential for Targeting Autophagy in ER+ Breast Cancer

While endocrine therapy remains the mainstay of treatment for ER-positive, HER2-negative breast cancer, tumor progression and disease recurrence limit the utility of current standards of care. While existing therapies may allow for a prolonged progression-free survival, however, the growth-arrested (essentially dormant) state of residual tumor cells is not permanent and is frequently a precursor to disease relapse. Tumor cells that escape dormancy and regain proliferative capacity also tend to acquire resistance to further therapies. The cellular process of autophagy has been implicated in the adaptation, survival, and reactivation of dormant cells. Autophagy is a cellular stress mechanism induced to maintain cellular homeostasis. Tumor cells often undergo therapy-induced autophagy which, in most contexts, is cytoprotective in function; however, depending on how the autophagy is regulated, it can also be non-protective, cytostatic, or cytotoxic. In this review, we explore the literature on the relationship(s) between endocrine therapies and autophagy. Moreover, we address the different functional roles of autophagy in response to these treatments, exploring the possibility of targeting autophagy as an adjuvant therapeutic modality together with endocrine therapies

<i>Porphyromonas gingivalis</i> Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

<div><p>Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of <i>Porphyromonas gingivalis</i> in human monocyte-derived DCs (MoDCs). We employed a panel of <i>P. gingivalis</i> isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+<i>P. gingivalis</i> strains by MoDCs resulted in lower intracellular killing and higher intracellular content of <i>P. gingivalis</i>. Moreover, Mfa1+<i>P. gingivalis</i> was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+<i>P. gingivalis</i> strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, <i>P. gingivalis</i> uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+<i>P. gingivalis</i> promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.</p></div

TLRs activation restores LC3-II expression and inhibits the growth of Mfa1⁺Pg within human MoDCs.

<p><b>A)</b> Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (<i>E. coli</i> LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour. <b>B)</b> Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&2 ligands (<i>E. coli</i> LPS and Pam3csk4) <b>C)</b> The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** <i>p</i><0.001).</p

DC-SIGN mRNA expression in MoDCs at 12 hours.

<p>DC-SIGN mRNA expression in MoDCs at 12 hours.</p

Pearson’s co-localization of Rab5 (red) and P. gingivalis (green) signals.

<p>Pearson’s co-localization of Rab5 (red) and P. gingivalis (green) signals.</p

Induction of autophagy impairs the survival of Mfa1⁺Pg strain within MoDCs.

<p><b>A)</b> Survival of <i>P. gingivalis</i> strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show <i>P. gingivalis</i> strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1⁺Pg, Pg381 and FimA⁺Pg survival within MoDCs are shown in figures <b>B, C and D</b> respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1⁺Pg, Pg381 and FimA⁺Pg) between treated (rapamycin) and untreated were significantly different overtime (<i>p-value</i> <0.=001).</p

Mfa1⁺Pg up-regulate the expression of DC-SIGN in human MoDCs.

<p><b>A)</b> DC-SIGN mRNA expression in <i>P. gingivalis</i>-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2^-(ΔΔCt) method. The statistical analysis was performed using the <i>t-test</i>, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* <i>p</i><0.001). <b>B)</b> Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1⁺Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1⁺Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. <b>C)</b> Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1⁺Pg and FimA⁺Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* <i>p</i><0.01).</p

Low LC3-II signals in human MoDCs infected with Mfa1⁺Pg.

<p><b>A)</b> Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1⁺Pg, FimA⁺Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of <i>P. gingivalis</i> and LC3-II showed in the right panels. <b>B)</b> Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons of three different experiments (* <i>p</i><0.001, # <i>p</i><0.01). <b>C)</b> Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* <i>p</i><0.001, # <i>p</i><0.01).</p