Mechanisms that Promote Adhesion Defects and Genomic Instability to Produce Invasive Prostate Cancer

Abstract

Localized prostate cancer (PCa) display instabilities in both tissue homeostasis and genome. Normal human prostate epithelium consists of two cell layers. Basal cells localize outsize the glands, adherent to extracellular matrix (ECM) through integrins, and providing contextual cues for ordered repopulation of the luminal cell layer. To date, histological and cytological differentiation of prostate tissue are the primary cues used clinically to diagnose prostate cancer. Prostate cancer arises from high-grade prostatic intraepithelial neoplasia (HG-PIN). Early HG-PIN and localized prostate cancer are characterized by loss of basal cell layer, luminal layer expansion, enlarged nuclei/nucleoli, and large-scale genomic instability while lacking recurrent specific driver mutations. Yet, the molecular bases for how the instabilities arise that result in loss of tissue architecture and large-scale genomic alterations during cancer is unknown. In this study, we explored the mechanisms promoting instabilities in tissue homeostasis through adhesion defects and genomic instability during prostate cancer initiation and progression. A model summarizing this study is provided below (Figure 0 1). In normal prostate glands, β4 integrin and its variant β4E, which heterodimerize with α6 integrin, function in forming strong cell-ECM adhesion structures to maintain homeostasis of normal prostate tissue architecture. During prostate cancer initiation and progression, α6β4 and basal cells are lost early, generating instability in tissue homeostasis and loss of tissue architecture. Meanwhile, the major microtubule organizing centers in the cell, called centrosomes, were significantly lost during localized prostate cancer progression. Centrosome number is critical in maintaining a stable genome in normal cells. Loss of centrosome generated large-scale genomic instabilities in non-tumorigenic cells and transformed them to display invasive PCa phenotype in vitro and in vivo. Together, defects in α6β4 integrin mediated adhesions generate instability in tissue homeostasis and centrosome loss give rise to an unstable genome. These instabilities promote the formation of invasive prostate cancer. A model for the study is summarized below in Figure 0 1. Integrin α6β4 and α6β1 are essential, dynamic adhesion receptors for laminin 332 found on epithelial cells. Integrin α6β4 is required for formation of strong cell-ECM adhesion structures and induced migration, which are coordinated by regions of the β4C cytoplasmic domain. Integrin β4E, a unique splice variant of β4, expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique 114 amino acids sequence instead of β4C’s canonical 1089 amino acids. We determined the distribution of α6β4E protein within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. We found α6β4E was localized within a subset of luminal cells. α6β4E expression was induced by 3D hanging drop culture conditions. Induced expression of β4E by a doxycycline inducible system activated Src. During induced migration, α6β4E expression was restricted to a subset of follower cells with increased cell-cell and cell-ECM resistance properties. α6β4E was present in ring-like patterns measuring ~ 1.75 x 0.72 microns containing actin and CD9 at a cell-ECM location. In contrast, α6β4C was found only within hemidesmosome-like structures containing BP180. We showed that Integrin α6β4E is an inducible, dynamic adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions. HG-PIN markers include loss of α6β4 integrin or its ligand, laminin-332, and budding of tumor clusters into laminin-511 rich stroma. In a 3D culture condition, normal human, stable isogenic prostate epithelial cell lines formed spheroids representative as normal prostate glands, while the reduction of α6β4C integrin modeled the invasive budding phenotype in spheroids formed from. Normal cells continuously spin in 3D culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells, and luminal cells that secrete PSA. Basal cells were optimally positioned relative to the laminin-332 layer. α6β4C defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. The centrosome is the major microtubule-organizing center in cells and is crucial in defining mitotic spindle poles and forming cilia. To understand mechanisms of early molecular alterations that resulted large scale genomic instability independent of driver mutations, we investigated centrosome number alterations during localized prostate cancer progression. We established a method to quantify centrosomes at a single-cell level in different types of human tissue samples. Using this method, we found centrosome loss - which has not been described in human cancer - was associated with PCa progression. Centrosome alteration can be induced by microenvironment conditions like hypoxia. Non-transformed basal cells display centrosome loss under hypoxia condition, while non-transformed luminal cells or cancer cells had centrosome amplification under the same condition. Centrosome loss in non-tumorigenic prostate epithelial cells, generated either by a small molecule inhibitor or genetically knockout of the essential centrosomal gene Plk4, resulted in mitotic error, and gave rise to aneuploid and multinucleated progeny. In addition, centrosome loss produced invasive budding with luminal differentiation in the 3D model described above. Consistent with our integrin β4 study, centrosome loss disrupted β4 distribution, generating spheroids with β4 localized intracellular instead of outside with cell-ECM. Furthermore, both transient or chronic centrosome loss transformed prostate epithelial cells which produced highly proliferative and poorly differentiated invasive tumors in mice. Our findings suggest that centrosome loss could create a cellular crisis with oncogenic potential in prostate epithelial cells.