Ryan W. Askeland

MiR-93 enhances angiogenesis and metastasis by targeting LATS2

Here we report that miR-93, a miRNA in the miR-106B~25 cluster, a paralog of the miR-17–92 cluster, was significantly upregulated in human breast carcinoma tissues. We stably expressed miR-93 in the MT-1 human breast carcinoma cell line and found that tumors formed by the miR-93 cells contained more blood vessels than those formed by the control cells. Co-culture experiments indicated that the MT-1 cells displayed a high activity of adhesion with endothelial cells and could form larger and more tube-like structures with endothelial cells. Lung metastasis assays were performed in a mouse metastatic model, and it was found that expression of miR-93 promoted tumor cell metastasis to lung tissue. In cell culture, expression of miR-93 enhanced cell survival and invasion. We examined the potential target that mediated miR-93’s effects and found that the large tumor suppressor, homology 2 (LATS2) was a target of miR-93. Higher levels of LATS2 were associated with cell death in the tumor mass. Silencing LATS2 expression promoted cell survival, tube formation and invasion, while ectopic expression of LATS2 decreased cell survival and invasion. These findings demonstrated that miR-93 promoted tumor angiogenesis and metastasis by suppressing LATS2 expression. Our results suggest that the inhibition of miR-93 function may be a feasible approach to repress tumor metastasis.

MicroRNA miR-24 enhances tumor invasion and metastasis by targeting PTPN9 and PTPRF to promote EGF signaling

Summary MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells

It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity

Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨm) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨm was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.

Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette–Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity

Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)‐10 down‐regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL‐10 signalling could enhance the BCG‐induced Th1 response and anti‐tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti‐IL‐10 receptor 1 monoclonal antibody (anti‐IL‐10R1 mAb) increased the interferon (IFN)‐γ to IL‐10 ratio in both splenocyte cultures and urine. Mice bearing luciferase‐expressing MB49 (MB49‐Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate‐buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti‐IL‐10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K‐ras oncogene) known to be expressed in MB49‐Luc cells. Induction of ras mutation‐specific IFN‐γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti‐IL‐10R1 mAb, induces enhanced anti‐tumour immunity that is protective against lung metastasis. Anti‐IL‐10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high‐risk patients.

Regulator of G protein signaling 6 is a novel suppressor of breast tumor initiation and progression.

Breast cancer is a large global health burden and the most frequently diagnosed malignancy in women worldwide. Here, we utilize RGS6(-/-) mice to interrogate the role of regulator of G protein signaling 6 (RGS6), localized to the ductal epithelium in mouse and human breast, as a novel tumor suppressor in vivo. RGS6(-/-) mice exhibit accelerated 7,12-dimethylbenza[α]anthracene (DMBA)-induced tumor initiation and progression, as well as decreased overall survival. Analysis of carcinogenic aberrations in the mammary glands of DMBA-treated mice revealed a failure of the DNA damage response concurrent with augmented oncogenesis in RGS6(-/-) animals. Furthermore, RGS6 suppressed cell growth induced by either human epidermal growth factor receptor 2 or estrogen receptor activation in both MCF-7 breast cancer cells and mammary epithelial cells (MECs). MECs isolated from RGS6(-/-) mice also showed a deficit in DMBA-induced ATM/p53 activation, reactive oxygen species generation and apoptosis confirming that RGS6 is required for effective activation of the DNA damage response in these cells, a critical countermeasure against carcinogen-mediated genotoxic stress. The ability of RGS6 to simultaneously enhance DNA-damage-induced apoptotic signaling and suppress oncogenic cell growth likely underlie the accelerated tumorigenesis and cellular transformation observed in DMBA-treated RGS6(-/-) mice and isolated MECs, respectively. Unsurprisingly, spontaneous tumor formation was also seen in old female RGS6(-/-) but not in wild-type mice. Our finding that RGS6 is downregulated in all human breast cancer subtypes independent of their molecular classification indicates that obtaining a means to restore the growth suppressive and pro-apoptotic actions of RGS6 in breast might be a viable means to treat a large spectrum of breast tumors.

Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy

Objective:We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer. Background:TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized. Methods:Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts. Results:Knockdown of TFAP2C or RET inhibited GDNF (glial cell line–derived neurotrophic factor)–mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts. Conclusions:ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.

Inhibition of RET increases the efficacy of antiestrogen and is a novel treatment strategy for luminal breast cancer.

Purpose: Recent findings suggest that combination treatment with antiestrogen and anti-RET may offer a novel treatment strategy in a subset of patients with breast cancer. We investigated the role of RET in potentiating the effects of antiestrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to affect extracellular signal–regulated kinase 1/2 (ERK1/2) activation in primary breast cancer. Experimental Design: Growth response, ERK1/2 activation, Ki-67, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling were assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment. Results: Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (P = 0.01) and hormone insensitive (P < 0.001) estrogen receptor α (ERα)-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (P < 0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared with single agent (P = 0.003), with tamoxifen-reducing proliferative index and vandetanib-inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (P < 0.001) in RET-positive tumors versus 14% (P = ns) in RET-negative tumors. Conclusions: Vandetanib potentiated the antigrowth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of antiestrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer. Clin Cancer Res; 20(8); 2115–25. ©2014 AACR.

RABL6A Promotes Oxaliplatin Resistance in Tumor Cells and Is a New Marker of Survival for Resected Pancreatic Ductal Adenocarcinoma Patients.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early recurrence following pancreatectomy, rapid progression, and chemoresistance. Novel prognostic and predictive biomarkers are urgently needed to both stratify patients for clinical trials and select patients for adjuvant therapy regimens. This study sought to determine the biological significance of RABL6A (RAB, member RAS oncogene family-like protein 6 isoform A), a novel pancreatic protein, in PDAC. Analyses of RABL6A protein expression in PDAC specimens from 73 patients who underwent pancreatic resection showed that RABL6A levels are altered in 74% of tumors relative to adjacent benign ductal epithelium. Undetectable RABL6A expression, found in 7% (5/73) of patients, correlated with improved overall survival (range 41 to 118 months with 3/5 patients still living), while patients with RABL6A expression had a worse outcome (range 3.3 to 100 months, median survival 20.3 months) (P = 0.0134). In agreement with those findings, RABL6A expression was increased in pancreatic cancer cell lines compared to normal pancreatic epithelial cells, and its knockdown inhibited pancreatic cancer cell proliferation and induced apoptosis. Moreover, RABL6A depletion selectively sensitized cells to oxaliplatin-induced arrest and death. This work reveals that RABL6A promotes the proliferation, survival, and oxaliplatin resistance of PDAC cells, whereas its loss is associated with extended survival in patients with resected PDAC. Such data suggest RABL6A is a novel biomarker of PDAC and potential target for anticancer therapy.

RABL6A Promotes G1–S Phase Progression and Pancreatic Neuroendocrine Tumor Cell Proliferation in an Rb1-Dependent Manner

Mechanisms of neuroendocrine tumor (NET) proliferation are poorly understood, and therapies that effectively control NET progression and metastatic disease are limited. We found amplification of a putative oncogene, RABL6A, in primary human pancreatic NETs (PNET) that correlated with high-level RABL6A protein expression. Consistent with those results, stable silencing of RABL6A in cultured BON-1 PNET cells revealed that it is essential for their proliferation and survival. Cells lacking RABL6A predominantly arrested in G1 phase with a moderate mitotic block. Pathway analysis of microarray data suggested activation of the p53 and retinoblastoma (Rb1) tumor-suppressor pathways in the arrested cells. Loss of p53 had no effect on the RABL6A knockdown phenotype, indicating that RABL6A functions independent of p53 in this setting. By comparison, Rb1 inactivation partially restored G1 to S phase progression in RABL6A-knockdown cells, although it was insufficient to override the mitotic arrest and cell death caused by RABL6A loss. Thus, RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET proliferation and development. This work identifies RABL6A as a novel negative regulator of Rb1 that is essential for PNET proliferation and survival. We suggest RABL6A is a new potential biomarker and target for anticancer therapy in PNET patients.