PKD3 localizes to the endolysosomal compartment and maintains lysosomal homeostasis in TNBC

Abstract

The Protein Kinase D (PKD) family (comprising PKD1, PKD2 and PKD3), is involved in various cellular processes including proliferation, protein transport, migration, secretion and stress response. Previous studies demonstrated that the members of the PKD family are mainly located at the trans-Golgi network, which, together with the highly conserved structure within the three isoforms, suggests isoform redundancy. However, an isoform-specific dysregulation of PKD expression has been found in different tumors, including breast cancer. While PKD1 and PKD2 are the main isoforms in normal tissue and non-invasive breast cancers, an isoform switch occurs in triple-negative breast cancer (TNBC), in which PKD1 is epigenetically silenced, PKD3 is overexpressed and PKD2 levels remain mainly unchanged. This indicates a tumor-suppressing role for PKD1 and a pro-oncogenic role for PKD3, suggesting isoform-specific functions. In particular, in TNBC PKD3 was shown to be necessary to maintain the stem cell population, as well as to support cell proliferation via preserving the integrity of the endolysosomal compartment. However, the underlying mechanism regulating PKD3 function remains unknown. Using a 3D-on top culture system to better resemble the stiffening of the matrix occurring during breast cancer progression, this thesis aimed to understand the molecular mechanism regulating stemness in a PKD3-dependent manner in TNBC cells. Given the major role of the PKD family in controlling constitutive secretion at the level of the Golgi complex, it was initially hypothesized that autocrine and paracrine signalling could mediate this function. However, mass spectrometry analysis of the PKD3-dependent secretome, together with the use of MDA-MB-231 cells-derived conditioned medium in non-malignant cells did not confirm this assumption. In fact, confocal microscopy studies revealed that PKD3 does not localize to the trans-Golgi network, which explains its marginal role in controlling secretion in TNBC cells. In contrast, PKD3 co-localized with Rab7, Lamp1 and CD63, indicating for the first time the localization of PKD3 to endolysosomes. Mechanistically, studies using quantitative image analysis demonstrated that PKD3 is necessary to maintain Rab7 localization to endolysosomes, as loss of PKD3 led to the redistribution of Rab7 to the cytosol, a characteristic associated with its inactive state. Moreover, this effect on Rab7 localization also correlated with an altered luminal pH, indicating that PKD3 is necessary to maintain acidification of endolysosomes. Consequently, it is shown that this novel role of PKD3 in preserving lysosomal homeostasis and function supports stemness in TNBC cell lines via modulating the Wnt pathway. Taken together, the results obtained in this work demonstrate a previously unrecognized role for PKD3 at the endolysosomal compartment and provide new insights into the isoform-specific functions of the PKD family in TNBC cells. Finally, in pursuit of a more specific mechanism, a potential PKD3 substrate at endolysosomal membranes is postulated, which could explain the observed effects on Rab7 cycling.