R-spondin-3 promotes proliferation and invasion of breast cancer cells independently of Wnt signaling.

We recently identified R-spondin-3 (RSPO3) as a novel driver of breast cancer associating with reduced patient survival, expanding its clinical value as potential therapeutic target that had been recognized mostly for colorectal cancer so far. (Pre)clinical studies exploring RSPO3 targeting in colorectal cancer approach this indirectly with Wnt inhibitors, or directly with anti-RSPO3 antibodies. Here, we address the clinical relevance of RSPO3 in breast cancer and provide insight in the oncogenic activities of RSPO3. Utilizing the RSPO3 breast cancer mouse model, we show that RSPO3 drives the aberrant expansion of luminal progenitor cells expressing cancer stem cell marker CD61, inducing proliferative, poorly differentiated and invasive tumors. Complementary studies with tumor organoids and human breast cancer cell lines demonstrate that RSPO3 consistently promotes the proliferation and invasion of breast cancer cells. Importantly, RSPO3 exerts these oncogenic effects independently of Wnt signaling, rejecting the therapeutic value of Wnt inhibitors in RSPO3-driven breast cancer. Instead, direct RSPO3 targeting effectively inhibited RSPO3-driven growth of breast cancer cells. Conclusively, our data indicate that RSPO3 exerts unfavorable oncogenic effects in breast cancer, enhancing proliferation and malignancy in a Wnt-independent fashion, proposing RSPO3 itself as a valuable therapeutic target in breast cancer.

Crosstalk between androgen receptor and WNT/ß-catenin signaling causes sex-specific adrenocortical hyperplasia in mice.

Female bias is highly prevalent in conditions such as adrenal cortex hyperplasia and neoplasia, but the reasons behind this phenomenon are poorly understood. In this study, we show that overexpression of the secreted WNT agonist R-spondin 1 (RSPO1) leads to ectopic activation of WNT/ß-catenin signaling and causes sex-specific adrenocortical hyperplasia in mice. Although female adrenals show ectopic proliferation, male adrenals display excessive immune system activation and cortical thinning. Using a combination of genetic manipulations and hormonal treatment, we show that gonadal androgens suppress ectopic proliferation in the adrenal cortex and determine the selective regulation of the WNT-related genes Axin2 and Wnt4. Notably, genetic removal of androgen receptor (AR) from adrenocortical cells restores the mitogenic effect of WNT/ß-catenin signaling. This is the first demonstration that AR activity in the adrenal cortex determines susceptibility to canonical WNT signaling-induced hyperplasia.

LGR6-dependent conditional inactivation of E-cadherin and p53 leads to invasive skin and mammary carcinomas in mice.

Tissue-specific inactivation of E-cadherin combined with tumor suppressor loss leads to invasive and metastatic cancers in mice. While epidermal E-cadherin loss in mice induces squamous cell carcinomas, inactivation of E-cadherin in the mammary gland leads to invasive lobular carcinoma. To further explore the carcinogenic consequences of cell-cell adhesion loss in these compartments, we developed a new conditional mouse model inactivating E-cadherin (Cdh1) and p53 (Trp53) simultaneously in cells expressing the leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), a putative epithelial stem cell marker in the skin and alveolar progenitor marker in the mammary gland. Compound Lgr6-CreERT2;Cdh1F;Trp53F female mice containing either heterozygous or homozygous Cdh1F alleles were bred, and Lgr6-driven Cre expression was activated in pre-puberal mice using tamoxifen. We observed that 41% of the mice (16/39) developed mostly invasive squamous-type skin carcinomas, but also a non-lobular mammary tumor was formed. In contrast to previous K14cre or WAPcre E-cadherin and p53 compound models, no significant differences were detected in the tumor-free survival of Lgr6-CreERT2 heterozygous Cdh1F/WT;Trp53F/F versus homozygous Cdh1F/F;Trp53F/F mice (778 versus 754 days, p=0.5). One Cdh1F homozygous mouse presented with lung metastasis that originated from a non-lobular and ERα negative invasive mammary gland carcinoma with squamous metaplasia. In total, 2/8 (25%) Cdh1F heterozygous and 3/12 (25%) Cdh1F homozygous mice developed metastases to lungs, liver, lymph nodes, or the gastro-intestinal tract. In conclusion, we show that inducible and conditional Lgr6-driven inactivation of E-cadherin and p53 in mice causes squamous cell carcinomas of the skin in approximately 40% of the mice and an occasional ductal-type mammary carcinoma after long latency periods.

Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer.

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.

The role of R-spondin proteins in cancer biology.

R-spondin (RSPO) proteins constitute a family of four secreted glycoproteins (RSPO1-4) that have appeared as multipotent signaling ligands. The best-known molecular function of RSPOs lie within their capacity to agonize the Wnt/ß-catenin signaling pathway. As RSPOs act upon cognate receptors LGR4/5/6 that are typically expressed by stem cells and progenitor cells, RSPO proteins importantly potentiate Wnt/ß-catenin signaling especially within these proliferative stem cell compartments. Since multiple organs express LGR4/5/6 receptors and RSPO ligands within their stem cell niches, RSPOs can exert an influential role in stem cell regulation throughout the body. Inherently, over the last decade a multitude of reports implicated the deregulation of RSPOs in cancer development. First, RSPO2 and RSPO3 gene fusions with concomitant enhanced expression have been identified in colon cancer patients, and proposed as an alternative driver of Wnt/ß-catenin hyperactivation that earmarks cancer in the colorectal tract. Moreover, the causal oncogenic capacity of RSPO3 overactivation has been demonstrated in the mouse intestine. As a paradigm organ in this field, most of current knowledge about RSPOs in cancer is derived from studies in the intestinal tract. However, RSPO gene fusions as well as enhanced RSPO expression have been reported in multiple additional cancer types, affecting different organs that involve divergent stem cell hierarchies. Importantly, the emerging oncogenic role of RSPO and its potential clinical utility as a therapeutic target have been recognized and investigated in preclinical and clinical settings. This review provides a survey of current knowledge on the role of RSPOs in cancer biology, addressing the different organs implicated, and of efforts made to explore intervention opportunities in cancer cases with RSPO overrepresentation, including the potential utilization of RSPO as novel therapeutic target itself.

Insertional mutagenesis in a HER2-positive breast cancer model reveals ERAS as a driver of cancer and therapy resistance.

Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS. The CIS most strongly associated with the ErbB2-transgenic genotype was the locus containing Eras (ES cell-expressed Ras), a constitutively active RAS-family GTPase. We show that upon expression, Eras acts as a potent oncogenic driver through hyperactivation of the PI3K/AKT pathway, in contrast to other RAS proteins that signal primarily via the MAPK/ERK pathway and require upstream activation or activating mutations to induce signaling. We additionally show that ERAS synergistically enhances HER2-induced tumorigenesis and, in this role, can functionally replace ERBB3/HER3 by acting as a more powerful activator of PI3K/AKT signaling. Although previously reported as pseudogene in humans, we observed ERAS RNA and protein expression in a substantial subset of human primary breast carcinomas. Importantly, we show that ERAS induces primary resistance to the widely used HER2-targeting drugs Trastuzumab (Herceptin) and Lapatinib (Tykerb/Tyverb) in vivo, and is involved in acquired resistance via selective upregulation during treatment in vitro, indicating that ERAS may serve as a novel clinical biomarker for PI3K/AKT pathway hyperactivation and HER2-targeted therapy resistance.

RSPO3 expands intestinal stem cell and niche compartments and drives tumorigenesis.

OBJECTIVE: The gross majority of colorectal cancer cases results from aberrant Wnt/ß-catenin signalling through adenomatous polyposis coli (APC) or CTNNB1 mutations. However, a subset of human colon tumours harbour, mutually exclusive with APC and CTNNB1 mutations, gene fusions in RSPO2 or RSPO3, leading to enhanced expression of these R-spondin genes. This suggested that RSPO activation can substitute for the most common mutations as an alternative driver for intestinal cancer. Involvement of RSPO3 in tumour growth was recently shown in RSPO3-fusion-positive xenograft models. The current study determines the extent into which solely a gain in RSPO3 actually functions as a driver of intestinal cancer in a direct, causal fashion, and addresses the in vivo activities of RSPO3 in parallel. DESIGN: We generated a conditional Rspo3 transgenic mouse model in which the Rspo3 transgene is expressed upon Cre activity. Cre is provided by cross-breeding with Lgr5-GFP-CreERT2 mice. RESULTS: Upon in vivo Rspo3 expression, mice rapidly developed extensive hyperplastic, adenomatous and adenocarcinomatous lesions throughout the intestine. RSPO3 induced the expansion of Lgr5+ stem cells, Paneth cells, non-Paneth cell label-retaining cells and Lgr4+ cells, thus promoting both intestinal stem cell and niche compartments. Wnt/ß-catenin signalling was modestly increased upon Rspo3 expression and mutant Kras synergised with Rspo3 in hyperplastic growth. CONCLUSIONS: We provide in vivo evidence that RSPO3 stimulates the crypt stem cell and niche compartments and drives rapid intestinal tumorigenesis. This establishes RSPO3 as a potent driver of intestinal cancer and proposes RSPO3 as a candidate target for therapy in patients with colorectal cancer harbouring RSPO3 fusions.

IRS4 induces mammary tumorigenesis and confers resistance to HER2-targeted therapy through constitutive PI3K/AKT-pathway hyperactivation.

In search of oncogenic drivers and mechanisms affecting therapy resistance in breast cancer, we identified Irs4, a poorly studied member of the insulin receptor substrate (IRS) family, as a mammary oncogene by insertional mutagenesis. Whereas normally silent in the postnatal mammary gland, IRS4 is found to be highly expressed in a subset of breast cancers. We show that Irs4 expression in mammary epithelial cells induces constitutive PI3K/AKT pathway hyperactivation, insulin/IGF1-independent cell proliferation, anchorage-independent growth and in vivo tumorigenesis. The constitutive PI3K/AKT pathway hyperactivation by IRS4 is unique to the IRS family and we identify the lack of a SHP2-binding domain in IRS4 as the molecular basis of this feature. Finally, we show that IRS4 and ERBB2/HER2 synergistically induce tumorigenesis and that IRS4-expression confers resistance to HER2-targeted therapy. Taken together, our findings present the cellular and molecular mechanisms of IRS4-induced tumorigenesis and establish IRS4 as an oncogenic driver and biomarker for therapy resistance in breast cancer.

Integrated ß-catenin, BMP, PTEN, and Notch signalling patterns the nephron.

The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in ß-catenin activity along the axis of the nephron tubule. By modifying ß-catenin activity, we force cells within nephrons to differentiate according to the imposed ß-catenin activity level, thereby causing spatial shifts in nephron segments. The ß-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises ß-catenin activity and promotes segment identities associated with low ß-catenin activity. ß-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating ß-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.

Wnt5a promotes human colon cancer cell migration and invasion but does not augment intestinal tumorigenesis in Apc1638N mice.

Whereas aberrant activation of canonical Wnt/ß-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.

Colorectal cancers choosing sides.

In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes focusing specifically on colorectal location. Combining this analysis with published data, we propose a mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in ß-catenin signalling.

ß-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis.

Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.