Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Activation of Canonical BMP4-SMAD7 Signaling Suppresses Breast Cancer Metastasis.

Metastasis is the major cause of death in patients with cancer; with no therapeutic cure, treatments remain largely palliative. As such, new targets and therapeutic strategies are urgently required. Here, we show that bone morphogenetic protein-4 (BMP4) blocks metastasis in animal models of breast cancer and predicts improved survival in patients. In preclinical models of spontaneous metastasis, BMP4 acted as an autocrine mediator to modulate a range of known metastasis-regulating genes, including Smad7, via activation of canonical BMP-SMAD signaling. Restored BMP4 expression or therapeutically administered BMP4 protein, blocked metastasis and increased survival by sensitizing cancer cells to anoikis, thereby reducing the number of circulating tumor cells. Gene silencing of Bmp4 or its downstream mediator Smad7, reversed this phenotype. Administration of recombinant BMP4 markedly reduced spontaneous metastasis to lung and bone. Elevated levels of BMP4 and SMAD7 were prognostic for improved recurrence-free survival and overall survival in patients with breast cancer, indicating the importance of canonical BMP4 signaling in the suppression of metastasis and highlighting new avenues for therapy against metastatic disease. SIGNIFICANCE: Targeting the BMP4-SMAD7 signaling axis presents a novel therapeutic strategy to combat metastatic breast cancer, a disease that has had no reduction in patient mortality over 20 years. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1304/F1.large.jpg.

Loss of GRHL3 leads to TARC/CCL17-mediated keratinocyte proliferation in the epidermis.

Identifying soluble factors that influence epidermal integrity is critical for the development of preventative and therapeutic strategies for disorders such as ichthyosis, psoriasis, dermatitis and epidermal cancers. The transcription factor Grainyhead-like 3 (GRHL3) is essential for maintaining barrier integrity and preventing development of cutaneous squamous cell carcinoma (SCC); however, how loss of this factor, which in the skin is expressed exclusively within suprabasal epidermal layers triggers proliferation of basal keratinocytes, had thus far remained elusive. Our present study identifies thymus and activation-regulated chemokine (TARC) as a novel soluble chemokine mediator of keratinocyte proliferation following loss of GRHL3. Knockdown of GRHL3 in human keratinocytes showed that of 42 cytokines examined, TARC was the only significantly upregulated chemokine. Mouse skin lacking Grhl3 presented an inflammatory response with hallmarks of TARC activation, including heightened induction of blood clotting, increased infiltration of mast cells and pro-inflammatory T cells, increased expression of the pro-proliferative/pro-inflammatory markers CD3 and pSTAT3, and significantly elevated basal keratinocyte proliferation. Treatment of skin cultures lacking Grhl3 with the broad spectrum anti-inflammatory 5-aminosalicylic acid (5ASA) partially restored epidermal differentiation, indicating that abnormal keratinocyte proliferation/differentiation balance is a key driver of barrier dysfunction following loss of Grhl3, and providing a promising therapeutic avenue in the treatment of GRHL3-mediated epidermal disorders.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Stage-dependent therapeutic efficacy in PI3K/mTOR-driven squamous cell carcinoma of the skin.

Cutaneous squamous cell carcinoma (SCC) is a recurrent cancer that is prevalent in predisposed subjects such as immunosuppressed patients and patients being treated for other malignancies. Model systems to trial therapies at different stages of SCC development are lacking, therefore precluding efficient therapeutic interventions. Here, we have disrupted the expression of the tumor suppressor GRHL3 to induce loss of PTEN and activation of the PI3K/mTOR signaling pathway in mice and human skin, promoting aggressive SCC development. We then examined the potential for targeting PI3K/mTOR and an oncogenic driver miR-21, alone and in combination, for the prevention and treatment of SCC during the initiation, promotion/progression and establishment stages. Treatment with PI3K/mTOR inhibitors completely prevented tumor initiation, and these inhibitors significantly delayed the course of papilloma progression to malignancy. However, established SCC did not undergo any growth regression, indicating that this therapy is ineffective in established cancers. Mechanistically, the resistant SCCs displayed increased miR-21 expression in mice and humans where antagonists of miR-21 rescued expression levels of GRHL3/PTEN, but the combination of miR-21 antagonism with PI3K/mTOR inhibition resulted in acquired SCC resistance in part via c-MYC and OCT-4 upregulation. In conclusion, our data provide molecular evidence for the efficacy of targeting oncogenic drivers of SCC during the initiation and promotion stages and indicate that combination therapy may induce an aggressive phenotype when applied in the establishment stage.

The RNA binding protein quaking regulates formation of circRNAs.

Circular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT. Furthermore, by modulating QKI levels, we show the effect on circRNA abundance is dependent on intronic QKI binding motifs. Critically, the addition of QKI motifs is sufficient to induce de novo circRNA formation from transcripts that are normally linearly spliced. These findings demonstrate circRNAs are both purposefully synthesized and regulated by cell-type specific mechanisms, suggesting they play specific biological roles in EMT.

The tyrosine phosphatase PTPN14 (Pez) inhibits metastasis by altering protein trafficking.

Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway. Knocking down PTPN14 in tumor cells or injecting the peritoneum of mice with conditioned medium from PTPN14-deficient cell cultures promoted the growth and metastasis of breast cancer xenografts. Loss of catalytically functional PTPN14 increased the secretion of growth factors and cytokines, such as IL-8 (interleukin-8), and increased the abundance of EGFR (epidermal growth factor receptor) at the cell surface of breast cancer cells and of FLT4 (vascular endothelial growth factor receptor 3) at the cell surface of primary lymphatic endothelial cells. We identified RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-δ) as binding partners and substrates of PTPN14. Similar to cells overexpressing PTPN14, receptor trafficking to the cell surface was inhibited in cells that lacked PRKCD or RIN1 or expressed a nonphosphorylatable RIN1 mutant, and cytokine secretion was decreased in cells treated with PRKCD inhibitors. Invasive breast cancer tissue had decreased expression of PTPN14, and patient survival was worse when tumors had increased expression of the genes encoding RIN1 or PRKCD. Thus, PTPN14 prevents metastasis by restricting the trafficking of both soluble and membrane-bound proteins.

Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is required for the specification of tissues during embryonic development and is recapitulated during the metastatic progression of tumors. The miR-200 family plays a critical role in enforcing the epithelial state with their expression lost in cells undergoing EMT. EMT can be mediated by activation of the ZEB1 and ZEB2 (ZEB) transcription factors, which repress miR-200 expression via a self-reinforcing double negative feedback loop to promote the mesenchymal state. However, it remains unclear what factors drive and maintain epithelial-specific expression of miR-200 in the absence of EMT-inducing factors. Here, we show that the transcription factor Specificity Protein 1 (Sp1) binds to the miR-200b∼200a∼429 proximal promoter and activates miR-200 expression in epithelial cells. In mesenchymal cells, Sp1 expression is maintained, but its ability to activate the miR-200 promoter is perturbed by ZEB-mediated repression. Reduction of Sp1 expression caused changes in EMT-associated markers in epithelial cells. Furthermore, we observed co-expression of Sp1 and miR-200 during mouse embryonic development wherein miR-200 expression was only lost in regions with high ZEB expression. Together, these findings indicate that miR-200 family members require Sp1 to drive basal expression and to maintain an epithelial state.

An autocrine TGF-beta/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. A double-negative feedback loop involving the miR-200 family and ZEB (zinc finger E-box-binding homeobox) transcription factors has been postulated to control the balance between epithelial and mesenchymal states. Here we demonstrate using the epithelial Madin Darby canine kidney cell line model that, although manipulation of the ZEB/miR-200 balance is able to repeatedly switch cells between epithelial and mesenchymal states, the induction and maintenance of a stable mesenchymal phenotype requires the establishment of autocrine transforming growth factor-ß (TGF-ß) signaling to drive sustained ZEB expression. Furthermore, we show that prolonged autocrine TGF-ß signaling induced reversible DNA methylation of the miR-200 loci with corresponding changes in miR-200 levels. Collectively, these findings demonstrate the existence of an autocrine TGF-ß/ZEB/miR-200 signaling network that regulates plasticity between epithelial and mesenchymal states. We find a strong correlation between ZEBs and TGF-ß and negative correlations between miR-200 and TGF-ß and between miR-200 and ZEBs, in invasive ductal carcinomas, consistent with an autocrine TGF-ß/ZEB/miR-200 signaling network being active in breast cancers.