Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

Therapy resistance remains a major barrier to improving outcomes in HER2-positive breast cancer, with dormant tumor cells (DTCs) and cancer stem cells (CSCs) playing critical roles in recurrence and treatment failure. Herein, we investigated the interplay between dormancy and CSCs features in HER2-amplified breast cancer cell models and evaluated the role of the JAK1–STAT3 axis in sustaining these therapy-resistant phenotypes. Using an in vitro dormancy model induced by HER2 inhibition, we observed a reversible quiescent state characterized by decreased proliferation and viability, accompanied by a significant increase in the CSC marker CD44. CD44 expression was rapidly induced following HER2 inhibition, preceding measurable effects on cell viability, and persisted throughout the dormancy phase. CD44-positive populations showed reduced sensitivity to HER2 inhibition and displayed robust proliferative recovery upon therapy withdrawal. Functional studies revealed that the inhibition of JAK1, but not STAT3, impaired the recovery of CD44-positive populations and decreased their proliferative capacity, suggesting a critical role for JAK1 in maintaining the CSC phenotype during therapy. These findings underscore the importance of CD44 as a marker and mediator of therapy resistance and suggest that targeting CD44-positive cells or the JAK1 signaling axis could improve the efficacy of HER2-targeted therapies. Our study provides novel insights into the mechanisms underlying dormancy and CSC induction in HER2-positive breast cancer and highlights potential strategies to mitigate therapy resistance and prevent disease recurrence.

Penile, vulvar and anal neoplasms show an incidence lower than 0.5% of the population per year and therefore can be considered as rare cancers but with a dramatic impact on quality of life and survival. This work describes the experience of a Chilean cancer center using multiplexed immunofluorescence to study a case series of four penile cancers, two anal cancers and one vulvar cancer and simultaneous detection of CD8, CD68, PD-L1, Cytokeratin and Ki-67 in FFPE samples. Fluorescent image analyses were performed using open sources for automated tissue segmentation and cell phenotyping. Our results showed an objective and reliable counting of objects with a single or combined labeling or within a specific tissue compartment. The variability was below 10%, and the correlation between analytical events was 0.92–0.97. Critical cell phenotypes, such as TILs, PD-L1+ or proliferative tumor cells were detected in a supervised and unsupervised manner with a limit of detection of less than 1% of relative abundance. Finally, the observed diversity and abundance of the different cell phenotypes within the tumor microenvironment for the three studied tumor types confirmed that our methodology is useful and robust to be applicable for many other solid tumors.

Ubiquitination regulates several biological processes. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1,187 genes of the human “ubiquitinome” using Amyloid Precursor Protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel key regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 caused a robust and rapid inhibition of Golgi-to-ER retrograde transport which leads to a potent blockage of macroautophagy by a mechanism associated with the retention of Atg9A and Rab1A at the Golgi apparatus. Because pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and their K-63-Ub chains, act as a crucial regulator factor for macroautophagy by controlling Golgi-to-ER retrograde transport.