Breast cancer is the globally most common malignancy amongst women. The incidence has increased dramatically over the last decades, particularly in the developing world. In 1980, 600 000 women were diagnosed with breast cancer, whereas 1.6 million received the same diagnosis in 2010 1. On the basis of these figures, it is obvious that preventive measurements are needed, combined with novel therapy strategies based on tumour biological characteristics. The mortality from breast cancer has decreased by around 30–40% since 1980s due to the use of adjuvant therapies and early detection. Targeted therapies such as trastuzumab (a monoclonal antibody that blocks HER2/neu receptor functions) and other anti-HER2-based drugs like lapatinib, pertuzumab and TDM-1 have lately been in focus. These drugs, in particular if used in combination together with chemotherapy, improve outcome in the metastatic setting. There is hope that improved use of these and other similar drugs may ultimately result in cure. The curative potential has already been demonstrated in the adjuvant and neoadjuvant settings. In the latter, histopathological remission is obtained in more than 50% of the cases when used in combination with chemotherapy. At the June 2012 Nobel conference ‘Breast cancer; progress and challenges in prevention, risk prediction, tumour biology and treatment’, world-leading experts gathered to discuss recent findings related to breast cancer biology and treatment. In addition to major support from the Nobel Assembly at Karolinska Institutet, the meeting was also sponsored by the Swedish Research Council, the Swedish Cancer Society, Journal of Internal Medicine (JIM) and the Karolinska Institutet-funded BRECT breast cancer research network. Some of the major and important topics are discussed in five review papers of this JIM issue. In this introductory paper, we address and introduce each of them. It is increasingly recognized that the tumour microenvironment exerts important regulatory functions that influence tumour growth and metastasis. Two reviews in this JIM issue address the complex roles of hypoxia and the importance of activation of stromal cells at metastatic sites 2, 3. The review by Rundqvist and Johnson describes how hypoxia induces functional alterations in various cells of the tumour microenvironment, including macrophages, vascular cells and fibroblasts 2. These responses affect metastasis through multiple mechanisms, including alterations in the paracrine signalling to malignant cells and changes in the functional characteristics of the vasculature or immune cells. The classical ‘seed-and-soil’ hypothesis of cancer metastasis emphasizes the importance of host cell–tumour cell interactions in metastasis. These ideas receive new support through a series of findings outlining the importance of host cell activation in metastatic seeding 3. A number of recent experimental studies suggest that the activation of such ‘pre-metastatic niches’ includes both recruitment of bone marrow–derived cells and activation of resident cells including fibroblast. Obviously, targeting of these processes is a highly interesting strategy for adjuvant cancer therapy. Important progress in the characterization of normal tissue stem cells, and their progenitors, has been made over the recent past. This is relevant because the phenotype of breast cancer cells is influenced both by the tumour cell of origin and the presence of specific somatic genetic alterations, including activation of oncogenes or inactivation of tumour suppressor genes. Moreover, the maintenance and spread of breast tumours is proposed to rely on a population(s) of tumour cells with self-renewal capacity and presenting several similarities to normal tissue stem cells. Martinez and Huelsken review in this JIM issue the present knowledge on the metastatic process and its dependency on cancer stem cells 4. Special emphasis is put on the understanding of which cancer cells are endowed with metastatic potential and the key role exerted by extracellular matrix (ECM) components in establishing cellular niches supporting the growth of metastatic cancer cells. Targeting such ECM proteins provides a new and interesting alternative therapeutic strategy to curb metastatic disease. Future breast cancer therapies will include improved selection of drugs, dosage and scheduling, based on a better understanding of breast cancer biology. Tumour heterogeneity is likely one of the most important factors limiting the efficacy of present therapies as described by Hayes and Paoletti 5. Another emerging issue is the clinically relevant disconcordance in marker expression between primary tumours and metastatic lesions. Endocrine therapies are cornerstones both in the early management of breast cancer as well as for recurrent disease 6. More recently, new targets have been identified including ‘mammalian target of rapamycin’ (mTOR). Inhibitors of this target together with endocrine therapies have demonstrated added value. Endocrine therapies can also be combined with anti-HER2-based therapies for the subgroup of oestrogen receptor–positive disease which also display alteration of the HER2 pathway. The effect so far is inferior to that obtained with the combined use of cytotoxic agents and anti-HER2-based therapies. During the metastatic spread, further genetic changes occur and act together with the tumour stroma. Monitoring the biology of metastatic disease is required for optimal treatment. Present procedures include different types of radiological investigations, including advanced functional imaging techniques. The additional need of biopsies of metastatic disease is becoming recognized, but the practical limitations are sometimes a problem. Circulation tumour cells and cell-free tumour DNA are emerging as highly interesting alternative sources of prognostic and response-predictive information 5. Targeted therapies have improved breast cancer patient survival, but still 5–20% of all breast cancer patients die from the disease within 10 years of initial diagnosis. At the same time, there is a large number of systemically treated women who are in no need of postsurgical adjuvant therapy. These women thus only experience the acute and late adverse health effects of systemic therapy. It is therefore of outmost importance to identify better prognostic markers and therapy predictors with the potential of reducing under- and overtreatment. From the reviews in this issue of JIM, we learn that tumour heterogeneity induces therapy resistance but that circulating tumour cells could be a novel way to monitor carcinogenic evolution. It is also recognized that to identify tumours that form metastasis, appropriate functional assessment of the primary disease is needed, that the microenvironment of the metastatic cells is of outmost important and that hypoxia influences the metastatic potential. Lastly, we learn that novel combinations of drugs, including endocrine drugs and drugs targeting mammalian target of rapamycin (mTOR) inhibitors, can be a way to increase therapeutic efficacy. These reviews all point at the complexity of carcinogenesis and the need of translational research efforts to identify markers of prognosis, therapy response and resistance. Bergh's research groups has received research grants from Astra-Zeneca (KI-AZ collaboration), Amgen,Bayer, Roche and Sanofi-Aventis for clinical studies including biopsy, gene expression and PET studies to the Karolinska Institutet and Karolinska University hospital, but no personal payments.

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis.We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufuf/f, Gli3Δ699, Wnt1-Cre, and Sox10NGFP mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging.We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity.We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU−GLI−SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Patients with the most common form of pancreatic cancer, ductal adenocarcinoma, barely respond to therapy. A study in mice now opens the door to a new approach. Olive et al.1 took aim at the tough layer of fibroblasts that encase such tumors and limit their accessibility to drugs. This inhibitor, in combination with a more conventional drug (gemcitabine), allowed blood vessels to penetrate such tumors and shrank them in a mouse model. The lives of the mice were extended only by a few weeks, but the approach opens the door to new ways of treating this difficult cancer.

The intestinal epithelium and the hair follicle represent examples of rapidly self-renewing tissue in adult mammals. We have recently identified a novel stem cell gene Lgr5 expressed in multiple adult tissues. At the bottoms of crypts in small intestine and colon as well as in hair follicles, Lgr5 marks cycling cells with stem cell properties (Barker et al. 2007; Jaks et al. 2008). Using an inducible Lgr5-Cre knockin allele in conjunction with the Rosa26-LacZ Cre reporter strain, long-term lineage-tracing experiments were performed in adult mice. The Lgr5(+ve) crypt-based cell generated all epithelial lineages during a 14-month period, implying that it represents the stem cell of the small intestine and colon. Similarly, lineage tracing during a 14-month period revealed that Lgr5(+ve) cells located in the bulge of the hair follicle sustained multiple rounds of hair growth. These observations support the counterintuitive notion that Lgr5(+ve) cells are actively cycling, yet represent long-term stem cells of these adult, self-renewing tissues.

Patched1 (PTCH1) is one of the key molecules involved in the Hedgehog (HH) signaling pathway and acts as the receptor of HH ligands. Additionally, PTCH1 inhibits the positive signal transductor Smoothened (SMO). Several PTCH1 splice variants are known but the functional differences among them are not clear. Here, we demonstrate the unique biological properties of the PTCH1 isoforms generated by alternative first exon usage. All isoforms examined worked as functional receptors of both Sonic HH and Desert HH. However, the signaling upregulated isoforms PTCH1-1B and -1C inhibited SMO and the pathway transcription factors glioma 1 (GLI1) and GLI2 to a higher extent than PTCH1-1 and -1Ckid. Moreover, in situ hybridizations allowed the detection of the Ptch1 isoforms in specific structures of the developing mouse embryo. Additionally, the differences in the N-terminal tail had a dramatic influence on the steady states of the proteins, with PTCH1-1B and -1C levels being significantly higher than PTCH1-1 and -1Ckid. This implies that the pronounced signaling inhibitory properties of PTCH1-1B and -1C may be mostly due to this high-protein expression rather than to intrinsic functional differences. Thus, our study supports a role of splicing variation and promoter choice for HH signaling regulation.

The developmentally important Hedgehog (Hh) signal transduction pathway, which has recently been implicated in several forms of cancer, is subject to regulation by several protein kinases. Here, we address the role of protein kinase C δ in pathway inhibition and show that cellular depletion or pharmacological inhibition of this kinase isoform results in a blockade of signalling between Suppressor of Fused and the Gli transcription factors. We further provide evidence that the observed pathway inhibition is independent of primary cilia and the mitogen-activated protein kinase kinase (Mek1) kinase. These findings allowed for the rapid dissection of downstream Hh pathway activation mechanisms in human tumour cells and demonstrate a surprising variation in how cells can activate signalling in a ligand- and receptor-independent manner.

Patched1 (PTCH1) is a human tumour suppressor that acts as an HH (Hedgehog) receptor protein and is important for embryonic patterning. PTCH1 mediates its effects through SMO (Smoothened) and represses the expression of HH target genes such as the transcription factor GLI1 (glioma 1) as well as PTCH1. Up-regulation of these genes has been observed in several cancer forms, including basal cell carcinoma, digestive track tumours and small cell lung cancer. The fact that PTCH1 down-regulates its own expression via 'negative feedback' is an important feature in HH signalling, as it keeps the balance between HH and PTCH1 activities that are essential for normal development. In the present study, we provide evidence that a novel mechanism allowing PTCH1 to maintain this balance may also exist. We show that gene activation by GLI1, the transcriptional effector of the pathway, can be down-regulated by PTCH1 without involvement of the canonical cascade of HH signalling events. Specifically, the SMO antagonist cyclopamine has no appreciable effects in blocking this PTCH1-mediated inhibition. Moreover, the negative GLI1 regulator SUFU (Suppressor of Fused) was also found to be dispensable. Additionally, deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180-786 and 1058-1210 are of highest significance in inhibiting GLI1 gene activation. This contrasts with the importance of the PTCH1 C-terminal domain for HH signalling.

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at approximately E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu(-/-) embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu(-/-) fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu(+/-) mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.

The naevoid basal cell carcinoma syndrome (NBCCS) is caused by mutations in the hedgehog receptor PTCH gene. It is characterized by developmental defects and a predisposition to the development of certain tumours, such as basal cell carcinoma, medulloblastoma and meningioma, and potentially fetal rhabdomyomas and embryonal rhabdomyosarcomas. This study aimed to analyse PTCH status in an NBCCS patient with fetal rhabdomyoma and to investigate whether deregulation of hedgehog signalling, as shown by altered expression of hedgehog pathway components and/or genetic imbalances, is a general finding in sporadic rhabdomyomas and rhabdomyosarcomas. The NBCCS patient had a novel PTCH germline mutation, 1370insT, and developed a fetal rhabdomyoma that harboured a 30 bp inframe deletion in the second allele resulting in homozygous inactivation of PTCH. Sporadic rhabdomyomas and rhabdomyosarcomas showed overexpression of PTCH (43/43) and GLI1 (41/43) mRNA, as determined by in situ hybridization, indicating ongoing active hedgehog signalling. Immunohistochemical staining revealed a subgroup of fetal rhabdomyomas and embryonal rhabdomyosarcomas (12/34) lacking PTCH immunoreactivity. Four of nine informative fetal rhabdomyomas and embryonal rhabdomyosarcomas showed loss of heterozygosity (LOH) in the PTCH region with two of these (one fetal rhabdomyoma and one embryonal rhabdomyosarcoma) also showing LOH in the SUFU region. These findings suggest that haploinsufficiency for the two tumour suppressor genes PTCH and SUFU, which are both active in the same signalling pathway, may be important for tumour development. Based on our results we propose that the pathogenesis of rhabdomyoblastic tumours, particularly fetal rhabdomyomas and embryonal rhabdomyosarcomas, involves deregulation of the hedgehog signalling pathway. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The PTCH1 tumor suppressor gene encodes a receptor for secreted hedgehog (HH) ligands and is important for proper proliferation, differentiation and patterning in almost every tissue and organ during embryogenesis. The PTCH1 protein works as a negative regulator of the HH-signaling pathway by repressing downstream signaling by the coreceptor smoothened (SMOH). Mutations in PTCH1 lead to constitutive expression of HH target genes and a relationship between mutated PTCH1 and the most common tumor form in the Western world, Basal Cell Carcinoma (BCC) has been clearly established. We here show that PTCH1 is transcriptionally regulated by three independent promoters generating transcripts with alternative first exons. We demonstrate that only one of two putative Gli-binding sites that were identified in the promoter region of PTCH1 is functional, and that the transactivating Gli proteins, GLI1, Gli2 and GLI3, bind and enhance transcription through this site. Moreover, a strong repression of both basal and induced PTCH1 transcription was observed following expression of a truncated version of GLI3. Most interestingly, the upstream components in the HH-signaling cascade, Sonic HH (SHH) and SMOH, solely operate through the functional Gli-binding site because mutation of the Gli-binding site resulted in the disappearance of the enhanced transcription induced by the Gli proteins, as well as by SHH or SMOH. This finding suggests that transcriptional activation of the PTCH1 gene mediated via the HH-signaling pathway is dependent on the single functional Gli-binding site.