Umeå University's logo

Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients.

TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling.

The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model.

In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner.

In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells.

In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.

Prostate cancer is the most common cancer in men, with the incidence rapidly increasing in Europe over the past two decades. Reliable biomarkers for prostate cancer are currently unavailable. Thus, there is an urgent need for improved biomarkers to diagnose prostate cancer at an early stage and to determine the best treatment options. Higher expression of transforming growth factor-β (TGFβ) has been reported in patients with aggressive cancer.

TGFβ is a multifunctional cytokine that acts as a tumor suppressor during early tumor development, and as a tumor promoter at later stages of cancer. TGFβ signals through the canonical Smad or non-Smad cascade via TGFβ type II and type I receptors. The TGFβ signaling cascade is regulated by various post-translational modifications of its key components. The present investigation aimed to identify a potential function of TRAF6 in TGFβ-induced responses in prostate cancer.

The first two articles of this thesis unveil the proteolytic cleavage of TGFβ type I receptor (TβRI), and the biological importance of the liberated TβRI intracellular domain (TβRI-ICD) in the nucleus. We found that tumor necrosis factor receptor-associated factor 6 (TRAF6) polyubiquitinates TβRI, which leads to cleavage of TβRI by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta (PKCζ)-dependent manner. Following ectodomain shedding, TβRI undergoes a second cleavage by presenilin 1 (PS1), which liberates TβRI-ICD. TβRI-ICD translocates to the nucleus, where it regulates its own expression as well as expression of the pro-invasive gene Snail1, thereby promoting invasion. We further found that TβRI-ICD associates with Notch intracellular domain (NICD) to drive expression of the pro-invasive gene Snail1, as well as Notch1 ligand Jag1.

The third article provides evidence that TRAF6 promotes Lys63-linked polyubiquitination of TβRI at Lys178 in a TGFβ-dependent manner. TβRI polyubiquitination was found to be a prerequisite for TβRI nuclear translocation, and thus for regulation of the genes involved in cell cycle, differentiation, and invasion of prostate cancer cells.

In the fourth article we investigated the role of the pro-invasive gene Snail1 in TGFβ-induced epithelial-to-mesenchymal transition (EMT) in prostate cancer cells.