No abstract

Although the introduction of novel targeted agents has improved patient outcomes in several human cancers, no such advance has been achieved in muscle-invasive bladder cancer (MIBC). However, recent sequencing efforts have begun to dissect the complex genomic landscape of MIBC, revealing distinct molecular subtypes and offering hope for implementation of targeted therapies. Her2 (ERBB2) is one of the most established therapeutic targets in breast and gastric cancer but agents targeting Her2 have not yet demonstrated anti-tumor activity in MIBC. Through an integrated analysis of 127 patients from three centers, we identified alterations of Her2 at the DNA, RNA and protein level, and demonstrate that Her2 relevance as a tumor driver likely may vary even within ERBB2 amplified cases. Importantly, tumors with a luminal molecular subtype have a significantly higher rate of Her2 alterations than those of the basal subtype, suggesting that Her2 activity is also associated with subtype status. Although some of our findings present rare events in bladder cancer, our study suggests that comprehensively assessing Her2 status in the context of tumor molecular subtype may help select MIBC patients most likely to respond to Her2 targeted therapy.

Transforming growth factor-b (TGF-b) signals through membrane-bound serine/threonine kinase receptors, which upon stimulation phosphorylate Smad proteins and thereby trigger their nuclear translocation and transcriptional activity. Although the three mammalian isoforms of TGF-b are highly homologous at the level of sequence, analysis of their in vivo function by gene knockouts revealed striking differences, suggesting no signi®cant functional redundancy between TGF-b1, -2 and -3. While signal transduction by TGF-b1 has been well characterized, receptor binding and activation by the TGF-b2 isoform is less well understood. Here, we show that TbRII-B, an alternatively spliced variant of the TGF-b type II receptor, is a TGF-b2 binding receptor, which mediates signalling via the Smad pathway in the absence of any TGF-b type III receptor (TbRIII). L6 cells lacking endogenous TbRIII as well as TbRII-B do not respond to TGF-b2. Transfection of these cells with TbRII-B restores TGF-b2 sensitivity. The expression of TbRII-B is restricted to cells originating from tissues such as bone where the isoform TGF-b2 has a predominant role. This re¯ects the importance of this receptor in TGF-b isoform-speci®c signalling.

The CCND1 gene encodes the protein CyclinD1, which is an important promoter of the cell cycle and a prognostic and predictive factor in different cancers. CCND1 is amplified to a substantial proportion in various tumors, and this may contribute to CyclinD1 overexpression. In bladder cancer, information about the clinical relevance of CCND1/CyclinD1 alterations is limited. In the present study, amplification status of CCND1 and expression of CyclinD1 were evaluated by fluorescence in situ hybridization and immunohistochemistry on tissue microarrays from 152 lymph node-positive urothelial bladder cancers (one sample each from the center and invasion front of the primary tumors, two samples per corresponding lymph node metastasis) treated by cystectomy and lymphadenectomy. CCND1 amplification status and the percentage of immunostained cancer cells were correlated with histopathological tumor characteristics, cancer-specific survival and response to adjuvant chemotherapy. CCND1 amplification in primary tumors was homogeneous in 15% and heterogeneous in 6% (metastases: 22 and 2%). Median nuclear CyclinD1 expression in amplified samples was similar in all tumor compartments (60-70% immunostained tumor nuclei) and significantly higher than in non-amplified samples (5-20% immunostained tumor nuclei; Po0.05). CCND1 status and CyclinD1 expression were not associated with primary tumor stage or lymph node tumor burden. CCND1 amplification in primary tumors (P ¼ 0.001) and metastases (P ¼ 0.02) and high nuclear CyclinD1 in metastases (P ¼ 0.01) predicted early cancerrelated death independently. Subgroup analyses showed that chemotherapy was particularly beneficial in patients with high nuclear CyclinD1 expression in the metastases, whereas expression in primary tumors and CCND1 status did not predict chemotherapeutic response. In conclusion, CCND1 amplification status and CyclinD1 expression are independent risk factors in metastasizing bladder cancer. High nuclear CyclinD1 expression in lymph node metastases predicts favorable response to chemotherapy. This information may help to personalize prognostication and administration of adjuvant chemotherapy.

Activin is a member of the transforming growth factor b (TGF-b) family, which plays a crucial role in skin morphogenesis and wound healing. To gain insight into the underlying mechanisms of action, we searched for activin-regulated genes in cultured keratinocytes. One of the identified target genes encodes Id1, a negative regulator of helix-loop-helix transcription factors. We show that Id1, Id2, and Id3 are strongly downregulated by activin in keratinocytes in vitro and in vivo. To determine the role of Id1 in keratinocyte biology, we generated stable HaCaT keratinocyte cell lines overexpressing this protein. Our results revealed that enhanced levels of Id1 do not affect proliferation of keratinocytes in monoculture under exponential culture conditions or in response to activin or TGF-b1. However, in three-dimensional organotypic cultures, Id1-overexpressing HaCaT cells formed a hyperthickened and disorganized epithelium that was characterized by enhanced keratinocyte proliferation, abnormal differentiation, and an increased rate of apoptosis. These results identify an important function of Id1 in the regulation of epidermal homeostasis.