Lineage plasticity has emerged as an important mechanism of treatment resistance in prostate cancer that results into multiple different tumor states. Recent classifications efforts have revealed that besides the androgen receptor pathway positive adenocarcinomas (CRPC-AD) and the terminal neuroendocrine tumors (NEPC), there are also tumors enriched in a “hybrid state” characterized by the expression of mesenchymal and stem cell traits. This phenotype is termed mesenchymal and stem-like prostate cancer (MSPC). The overarching goal of this application is to identify novel molecular mechanisms underlying the MSPC phenotype that can be targeted therapeutically. Our hypothesis is that the loss of PKCλ/ ι (encoded by PRKCI gene), by upregulating the CD44/Hyaluronan (HA) axis, induces the mesenchymal phenotype characterized by the activation of epithelial EMT and the generation of desmoplastic tumor stroma, both critical events for tumor progression and therapy resistance. We postulate that the detailed characterization of the mechanisms whereby PKCλ/ ι controls the CD44/HA axis will provide critical new information on the molecular pathways leading to MSPC progression and will offer a rationale for the selection of patients susceptible to respond to new therapies designed at mesenchymal/stromal targets. Our preliminary data demonstrate that (1) human MSPC has low PRKCI levels and a highly desmoplastic stroma; (2) PKCλ/ ι loss in the prostate epithelium in vivo induces EMT and promotes a desmoplastic response that remodels the tumor microenvironment; (3) Low PRKCI levels correlate with upregulation of the CD44/HA axis in human PCa, supporting the human relevance of these findings. Based on these premises, in this proposal we will address the following specific Aims: Aim 1A: Determine how interfering with CD44 or its ligands (OPN/SPP1, HA) block MSPC and enzalutamide resistance promoted by PKCλ/ ι- deficiency in the epithelium; Aim 1B: Determine the role of the CD44/HA axis in stromal activation and the stromal-epithelial feedback crosstalk; Aim 1C: Determine the molecular mechanism whereby PKCλ/ ι regulates CD44; Aim 2A: Determine the role of prostate epithelial CD44 in driving MSPC in vivo; Aim 2B: Determine the role of prostate stromal CD44 in driving MSPC in vivo; Aim 3A: Determine the therapeutic potential of PEGPH20 in combination with enzalutamide in the treatment of MSPC tumors and Aim 3B: Investigate its mechanism of action at a single-cell level. The results of this proposal will contribute to a more comprehensive understanding of the mechanisms driving the MSPC type of PCa and will be key for the design of new more selective and effective therapies for this type of aggressive neoplasia.