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Metastatic castration-resistant prostate cancer (mCRPC) remains the primary cause of prostate cancer-related mortality. Despite the availability of treatments, the molecular mechanisms underlying tumor invasion and metastasis are not fully understood, highlighting the need for novel therapeutic strategies. In this study, we developed fully human monoclonal antibodies (mAbs) that prevent the proteolytic cleavage of the transforming growth factor-beta (TGFβ) type I receptor (TβRI) by steric hindrance. This cleavage, mediated by the metalloprotease ADAM17 (a disintegrin and metalloprotease domain 17; also known as TACE), results in the generation of a soluble intracellular domain (TβRI-ICD) that is translocated to the nucleus of castration-resistant prostate cancer (CRPC) cells and promotes epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. High levels of TGFBR1 correlated with poor survival in two independent clinical cohorts of patients with mCRPC, and a strong positive correlation between TGFBR1 and ADAM17 expression was observed. In a preclinical human orthotopic mCRPC mouse model, treatment with therapeutic mAbs effectively prevented the nuclear accumulation of TβRI-ICD, inhibited EMT, and suppressed tumor growth, invasion, and metastasis. Notably, the therapeutic effect was comparable to that of docetaxel, a current standard-of-care chemotherapy, without noticeable side effects on body weight, proximal aorta or heart function detected in immune-deficient mice. These findings suggest that targeting TβRI cleavage using specific mAbs is a novel precision medicine approach for the treatment of mCRPC. By selectively blocking the prometastatic activity of TβRI-ICD without disrupting physiological TGFβ signaling, this strategy may provide a safer and more effective alternative to existing therapies for advanced prostate cancer.

TGFβ functions as a tumor suppressor or promoter, depending on the context, making TGFβ a useful predictive biomarker. Genes related to TGFβ signaling and Aurora kinase were tested for their ability to predict the progression risk of primary prostate tumors. Using data from The Cancer Genome Atlas (TCGA), we trained an elastic-net regularized Cox regression model including a minimal set of gene expression, copy number (CN), and clinical data. A multi-step feature selection and regularization scheme was applied to minimize the number of features while maintaining predictive power. An independent hold-out cohort was used to validate the model. Expanding from prostate cancer, predictive models were similarly trained on all other eligible cancer types in TCGA. AURKA, AURKB, and KIF23 were predictive biomarkers of prostate cancer progression, and upregulation of these genes was associated with promotion of cell-cycle progression. Extending the analysis to other TCGA cancer types revealed a trend of increased predictive performance on validation data when clinical features were complemented with molecular features, with notable variation between cancer types and clinical endpoints. Our findings suggest that TGFβ signaling genes, prostate cancer related genes and Aurora kinases are strong candidates for patient-specific clinical predictions and could help guide personalized therapeutic decisions.

Background: Transforming growth factor β (TGFβ) is overexpressed in several advanced cancer types and promotes tumor progression. We have reported that the intracellular domain (ICD) of TGFβ receptor (TβR) I is cleaved by proteolytic enzymes in cancer cells, and then translocated to the nucleus in a manner dependent on the endosomal adaptor proteins APPL1/2, driving an invasiveness program. How cancer cells evade TGFβ-induced growth inhibition is unclear.

Methods: We performed microarray analysis to search for genes regulated by APPL1/2 proteins in castration-resistant prostate cancer (CRPC) cells. We investigated the role of TβRI and TRAF6 in mitosis in cancer cell lines cultured in 10% FBS in the absence of exogenous TGFβ. The molecular mechanism of the ubiquitination of AURKB by TRAF6 in mitosis and the formation of AURKB–TβRI complex in cancer cell lines and tissue microarrays was also studied.

Findings: During mitosis and cytokinesis, AURKB–TβRI complexes formed in midbodies in CRPC and KELLY neuroblastoma cells. TRAF6 induced polyubiquitination of AURKB on K85 and K87, protruding on the surface of AURKB to facilitate its activation. AURKB–TβRI complexes in patient's tumor tissue sections correlated with the malignancy of prostate cancer.

Interpretation: The AURKB–TβRI complex may become a prognostic biomarker for patients with risk of developing aggressive PC.

Transforming growth factor β (TGF-β) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-β to control TGF-β signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-β induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information. 

Cellular blebbing, caused by local alterations in cellsurface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrinindependent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension- mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol- interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

TGF-β signaling stimulates various intracellular pathways that can promote migration in tumor cells. These pathways are generally thought to be either dependent or independent of transcription factors called SMADs. One of the SMAD-independent pathways (PI3K-AKT) is mediated by a direct interaction between PI3K and the TGF-β type I receptor. However, Hamidi et al. found that the TGF-β–induced activation of PI3K depends on another ubiquitin ligase–mediated mechanism and a SMAD protein but is independent of the kinase function of TβRI. The binding of TGF-β to its receptor triggered the recruitment of PI3K and the ubiquitin ligase TRAF6, which polyubiquitylated the regulatory PI3K subunit p85α, thus enabling phosphorylation of the catalytic PI3K subunit p110, but only in the presence of SMAD7. The abundance of ubiquitylated p85α correlated with migration in cultured cells and prostate tumor grade in patient samples. TRAF6 mediates activation of the other “SMAD-independent” (JNK) pathway. These data suggest that, although distinct, the TGF-β signaling pathways are not as insulated from each other as was once thought.

The multifunctional cytokine transforming growth factor-beta (TGF beta) is produced by several types of cancers, including prostate cancer, and promote tumour progression in autocrine and paracrine manners. In response to ligand binding, the TGF beta type I receptor (T beta RI) activates Smad and non-Smad signalling pathways. The ubiquitin-ligase tumour necrosis factor receptor-associated factor 6 (TRAF6) was recently linked to regulate intramembrane proteolytic cleavage of the T beta RI in cancer cells. Subsequently, the intracellular domain (ICD) of T beta RI enters in an unknown manner into the nucleus, where it promotes the transcription of pro-invasive genes, such as MMP2 and MMP9. Here we show that the endocytic adaptor molecules APPL1 and APPL2 are required for TGF beta-induced nuclear translocation of T beta RI-ICD and for cancer cell invasiveness of human prostate and breast cancer cell lines. Moreover, APPL proteins were found to be expressed at high levels in aggressive prostate cancer tissues, and to be associated with T beta RI in a TRAF6-dependent manner. Our results suggest that the APPL-T beta RI complex promotes prostate tumour progression, and may serve as a prognostic marker.

Prostate cancer is the second leading cause of cancer-related death in men in the Western world. Deregulation of transforming growth factor β (TGFβ) signaling pathway is frequently detected in prostate cancer and contributes to tumor growth, migration, and invasion. In normal tissue and the early stages of cancer, TGFβ acts as a tumor suppressor by regulating proliferation, differentiation, and apoptosis. In later stages of cancer, TGFβ acts as a tumor promoter by inducing angiogenesis, tumor invasion, and migration. Thus, it is important to investigate the molecular mechanisms behind the tumor-promoting effects of TGFβ, which is the topic of this thesis.

The tumor necrosis factor receptor–associated factor 6 (TRAF6) controls non-canonical TGFβ signals due to its enzymatic activity, causing polyubiquitination of the cell membrane–bound, serine/threonine kinase TGFβ type I receptor (TβRI) and its subsequent cleavage in the extracellular domain by tumor necrosis factor a–converting enzyme (TACE) in a protein kinase C ζ (PKCζ)-dependent manner. TRAF6 also recruits the active g-secretase complex to the TβRI, resulting in a second cleavage in the transmembrane region and the liberation of the TβRI intracellular domain (TβRI-ICD), which enters the nucleus, where it associates with the transcriptional co-regulator p300. In Paper I, the aim was to elucidate by which mechanisms TβRI-ICD enters the nucleus. We found that the endocytic adaptor protein APPL1 interacts with TβRI and PKCζ. APPL proteins are required for TβRI translocation from endosomes to the nucleus via microtubules in a TRAF6-dependent manner. Moreover, APPL proteins are important for TGFβ-induced cell invasion, and high levels of APPL1 are detected by immunohistochemistry in prostate cancer. Finally, we demonstrated that the APPL1–TβRI complex visualized with the in situ proximity ligation assay (PLA) correlates with Gleason score, indicating that it might be a novel prognostic marker for aggressive prostate cancer. In Paper II, the aim was to explore by which mechanisms TGFβ causes activation of the AKT pathway, which regulates migration and therapy resistance of cancer cells. We found that the E3 ligase activity of TRAF6 induces Lys63-linked polyubiquitination of p85α upon TGFβ stimulation, resulting in plasma membrane recruitment, Lys63-linked polyubiquitination, and subsequent activation of AKT. Moreover, the TRAF6 and PI3K/AKT pathway were found to be crucial for the TGFβ-induced migration. Importantly, we demonstrated, by PLA, a correlation between Lys63-linked polyubiquitination of p85α and aggressive prostate cancer in tissue sections from patients with prostate cancer. In Paper III, the aim was to investigate the mechanisms for TGFβ-induced activation of PKCζ and the role of PKCζ in tumor regression. We found that TRAF6 caused Lys63-linked polyubiquitination of PKCζ. By using two novel chemical compounds that inhibit PKCζ, we demonstrated that PKCζ is crucial for prostate cancer cell survival and invasion. In Paper IV, the aim was to investigate further the target genes for the nuclear TβRI-ICD-APPL1 complex identified in Paper I. We provide evidence that APPL proteins and the TGFβ signaling pathway are important for cell proliferation. In summary, the results reported in this thesis suggest the potential usefulness of the identified signaling components of the tumor-promoting effects of TGFβ as drug targets and biomarkers for aggressive prostate cancer. 

The cytokine transforming growth factor b(TGFb) suppressescell proliferationand promotesapoptosis1. It signalsvia specific serine/threonine kinase receptors, i.e.TGFbtype I (TbRI) and type II (TbRII) receptors2,3,causing growth arrest of normal epithelial cells. However, TGFbis often overexpressed inadvanced cancers,and promotes proliferation of tumour cells and their invasion. The intracellular domain (ICD) of TbRI is cleaved offin cancer cells,and is translocated to the nucleus in an APPL1/2-dependent manner, drivingan invasiveness program4.The specific mechanism(s) whereby cancer cells escape pro-apoptotic signals induced by TGFbremainspoorly understood. Here, we report that TbRI and APPL1/2 proteins orchestrate this escape via the pro-survival protein survivin and Aurora kinase B (AURKB), a key regulatorof mitosis and chromosomal stability5. We show that TbRI and APPL1/2 control expression of AURKB and that TbRI-ICDand AURKB form a complex during the telophase in PC-3Uprostate cancerand KELLY neuroblastomacells. APPL1/2 and TbRI also form a complex with survivin, a pro-survival protein. The identified TbRI–AURKB-survivinpathwayrepresents a novel function for TbRI to promote survival and cell division of cancer cells.

One of the hallmarks for aggressivecancer is the capability oftumor cells to become invasive and metastatic. Cancer cells and tumor stromal cells oftenproduce high levels of transforming growth factor b(TGFb) which initiates intracellular signaling pathways in cancer cells in a contextualdependentmanner. Atypical protein kinase C z(PKCz) is a multifunctional protein which maintains cell polarity of normal epithelial cells, while itsaberrantexpression and activation is linked to tumor progression. Tumor necrosisfactor receptor-associated factor6 (TRAF6) is amplified in lung cancer and caninitiate intracellular oncogenic signals. In prostate cancer cellsTRAF6 promotesligand-induced proteolytic cleavage of TGFbtype I receptor(TbRI), and nuclear translocation of its intracellular domain (ICD) to confer invasion of cancer cells. Here we report our novel findingsthat PKCzharboursa TRAF6 consensus binding site and that TRAF6 causes Lys63-linked polyubiquitination of PKCz. TGFb-induced phosphorylationof PKCzis dependent on TRAF6in prostate cancer cells and we have investigated the potential usefulness of twodifferent inhibitors of PKCzas potential novel anti-cancer drugs.