Anti-LAMP2 monoclonal antibody, clone I5C5 [PE]

Purpose: Autophagy plays a critical role in PCa development. DAXX has a potent pro-survival effect by enhancing cell growth in PCa via suppression of autophagy. Here, we depicted a network governed by DAXX and SPOP by which the autophagy pathway is suppressed through the ubiquitination and modulation of key cellular signaling pathways mediators including LAMP2 and RARRES1. Materials and Methods: Through network-based bioinformatics approaches, the expression levels of DAXX, RARRES1, LAMP2, and SPOP genes was assessed in 50 PCa tissues and 50 normal adjacent from the same sample as well as 50 benign prostatic hyperplasia (BPH) tissues by quantitative RT-PCR. The normal adjacent tissues were taken from regions more than 5mm away from the bulk of those tumor tissues with clearly distinct margins. RNA extraction, cDNA synthesis and Real-time Quantitative RT-PCR were done for assessment of gene expression. To evaluate the primary gene network centered on autophagy pathway, according to the Query-dependent weighting algorithm, these two networks were integrated with Cytoscape 3.4 software. Results: We found that in PCa tissues the DAXX expression level was significantly increased (P < 0.001) and the expressions of SPOP, RARRES1, and LAMP2 were significantly down-regulated, when compared to both control groups including normal adjacent and BPH tissues. Moreover, significant correlations were observed between expression levels of all four genes. Additionally, ROC curve analysis revealed that LAMP2 had the most sensitivity and specificity. Conclusion: These findings suggest that the contribution of SPOP, DAXX, RARRES1, and LAMP2 together could be a putative regulatory element acting as a prognostic signature and therapeutic target in PCa.

Triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. To discover novel approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for cytotoxicity in TNBC cells. Only LT-IIc significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effect on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER+ cells, or SKBR-3 cells, HER2(+) cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as seen by accumulation of LC3B-II and p62. Morphologically, LT-IIc induced the formation of enlarged LAMP2+ autolysosomes, which was blocked by co-treatment with bafilomycin A1. LT-IIc induced apoptosis as demonstrated by the increase in caspase 3/7 activity and Annexin V staining. Co-treatment with necrostatin-1, however, demonstrated that the lethal response of LT-IIc is elicited, in part, by concomitant induction of necroptosis. Knockdown of ATG-5 failed to rescue LT-IIc-induced cytotoxicity, suggesting LT-IIc can exert its cytotoxic effects downstream or independently of autophagophore initiation. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy, while simultaneously blocking autolysosomal progression in TNBC cells, inducing a specific cytotoxicity in this breast cancer subtype.