Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis.

For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.

Pleiotropic tumor suppressor functions of WWOX antagonize metastasis.

Tumor progression and metastasis are the major causes of death among cancer associated mortality. Metastatic cells acquire features of migration and invasion and usually undergo epithelia-mesenchymal transition (EMT). Acquirement of these various hallmarks rely on different cellular pathways, including TGF-ß and Wnt signaling. Recently, we reported that WW domain-containing oxidoreductase (WWOX) acts as a tumor suppressor and has anti-metastatic activities involving regulation of several key microRNAs (miRNAs) in triple-negative breast cancer (TNBC). Here, we report that WWOX restoration in highly metastatic MDA-MB435S cancer cells alters mRNA expression profiles; further, WWOX interacts with various proteins to exert its tumor suppressor function. Careful alignment and analysis of gene and miRNA expression in these cells revealed profound changes in cellular pathways mediating adhesion, invasion and motility. We further demonstrate that WWOX, through regulation of miR-146a levels, regulates SMAD3, which is a member of the TGF-ß signaling pathway. Moreover, proteomic analysis of WWOX partners revealed regulation of the Wnt-signaling activation through physical interaction with Disheveled. Altogether, these findings underscore a significant role for WWOX in antagonizing metastasis, further highlighting its role and therapeutic potential in suppressing tumor progression.

AntimiR-155 Cyclic Peptide-PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity.

Efficient delivery of nucleic acids into cells still remains a great challenge. Peptide nucleic acids (PNAs) are DNA analogues with a neutral backbone and are synthesized by solid phase peptide chemistry. This allows a straightforward synthetic route to introduce a linear short peptide (a.k.a. cell-penetrating peptide) to the PNA molecule as a means of facilitating cellular uptake of PNAs. Herein, we have devised a synthetic route in which a cyclic peptide is prepared on a solid support and is extended with the PNA molecule, where all syntheses are accomplished on the solid phase. This allows the conjugation of the cyclic peptide to the PNA molecule with the need of only one purification step after the cyclic peptide-PNA conjugate (C9-PNA) is cleaved from the solid support. The PNA sequence chosen is an antimiR-155 molecule that is complementary to mature miR-155, a well-established oncogenic miRNA. By labeling C9-PNA with fluorescein isothiocyanate, we observe efficient cellular uptake into glioblastoma cells (U87MG) at a low concentration (0.5 µM), as corroborated by fluorescence-activated cell sorting (FACS) analysis and confocal microscopy. FACS analysis also suggests an uptake mechanism that is energy-dependent. Finally, the antimiR activity of C9-PNA was shown by analyzing miR155 levels by quantitative reverse transcription polymerase chain reaction and by observing a reduction in cell viability and proliferation in U87MG cells, as corroborated by XTT and colony formation assays. Given the added biological stability of cyclic versus linear peptides, this synthetic approach may be a useful and straightforward approach to synthesize cyclic peptide-PNA conjugates.

MiR-16-1-3p and miR-16-2-3p possess strong tumor suppressive and antimetastatic properties in osteosarcoma.

Osteosarcoma (OS) is an aggressive malignancy affecting mostly children and adolescents. MicroRNAs (miRNAs) play important roles in OS development and progression. Here we found that miR-16-1-3p and miR-16-2-3p "passenger" strands, as well as the "lead" miR-16-5p strand, are frequently downregulated and possess strong tumor suppressive functions in human OS. Furthermore, we report different although strongly overlapping functions for miR-16-1-3p and miR-16-2-3p in OS cells. Ectopic expression of these miRNAs affected primary tumor growth, metastasis seeding and chemoresistance and invasiveness of human OS cells. Loss-of-function experiments verified tumor suppressive functions of these miRNAs at endogenous levels of expression. Using RNA immunoprecipitation (RIP) assays, we identify direct targets of miR-16-1-3p and miR-16-2-3p in OS cells. Moreover, validation experiments identified FGFR2 as a direct target for miR-16-1-3p and miR-16-2-3p. Overall, our findings underscore the importance of passenger strand miRNAs, at least some, in osteosarcomagenesis.

WWOX Inhibits Metastasis of Triple-Negative Breast Cancer Cells via Modulation of miRNAs.

Triple-negative breast cancer (TNBC) is a heterogeneous, highly aggressive, and difficult to treat tumor type. The tumor suppressor WWOX spans FRA16D, a common fragile site that is commonly altered in breast cancer. Despite recent progress, the role of WWOX in TNBC metastasis is unknown. Here we report that WWOX inactivation correlates with advanced stages of TNBC and that its levels are frequently altered in TNBC cells. Ectopic restoration of WWOX in WWOX-negative TNBC cells inhibited metastasis while its depletion in WWOX-positive TNBC cells promoted metastasis. WWOX was a negative regulator of c-MYC, which regulated miR-146a expression and consequently fibronectin levels, contributing to an epithelial status of the cell. Treatment of TNBC cells with anti-miR-146a rescued the WWOX antimetastatic phenotype. Moreover, overexpression of MYC in WWOX-expressing TNBC cells overrode WWOX effects on miR-146a and fibronectin levels. Altogether, our data uncover an essential role for WWOX in antagonizing TNBC progression and highlight its potential use as a biomarker for metastasis. SIGNIFICANCE: These findings highlight the mechanism by which the tumor suppressor WWOX regulates metastasis of triple-negative breast cancer.See related commentary by Sharma, p. 1746.

Microenvironmental Cues Determine Tumor Cell Susceptibility to Neutrophil Cytotoxicity.

We have recently shown that neutrophil antitumor cytotoxicity is Ca2+ dependent and is mediated by TRPM2, an H2O2-dependent Ca2+ channel. However, neutrophil antitumor activity is dependent on context and is manifested in the premetastatic niche, but not at the primary site. We therefore hypothesized that expression of TRPM2 and the consequent susceptibility to neutrophil cytotoxicity may be associated with the epithelial/mesenchymal cellular state. We found that TRPM2 expression was upregulated during epithelial-to-mesenchymal transition (EMT), and mesenchymal cells were more susceptible to neutrophil cytotoxicity. Conversely, cells undergoing mesenchymal-to-epithelial transition (MET) expressed reduced levels of TRPM2, rendering them resistant to neutrophil cytotoxicity. Cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung. These data identify TRPM2 as the link between environmental cues at the primary tumor site, tumor cell susceptibility to neutrophil cytotoxicity, and disease progression. Furthermore, these data identify EMT as a process enhancing tumor-cell immune susceptibility and, by contrast, MET as a novel mode of immune evasion.Significance: EMT is required for metastatic spread and concomitantly enhances tumor cell susceptibility to neutrophil cytotoxicity. Cancer Res; 78(17); 5050-9. ©2018 AACR.

TRPM2 Mediates Neutrophil Killing of Disseminated Tumor Cells.

Neutrophils play a critical role in cancer, with both protumor and antitumor neutrophil subpopulations reported. The antitumor neutrophil subpopulation has the capacity to kill tumor cells and limit metastatic spread, yet not all tumor cells are equally susceptible to neutrophil cytotoxicity. Because cells that evade neutrophils have greater chances of forming metastases, we explored the mechanism neutrophils use to kill tumor cells. Neutrophil cytotoxicity was previously shown to be mediated by secretion of H2O2 We report here that neutrophil cytotoxicity is Ca2+ dependent and is mediated by TRPM2, a ubiquitously expressed H2O2-dependent Ca2+ channel. Perturbing TRPM2 expression limited tumor cell proliferation, leading to attenuated tumor growth. Concomitantly, cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung.Significance: These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. Cancer Res; 78(10); 2680-90. ©2018 AACR.

Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism.

Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation. We present here a combination of RNA inhibition and CRISPR/cas9 methods to identify possible off targets as well as potential compensatory effects. This approach is demonstrated by testing a possible role for Sema4B in glioma biology, in which our results implicate Sema4B as having a critical function. In stark contrast, by using shRNA over CRISPR/cas9 combined methodology, we clearly demonstrate that the Sema4B targeted shRNA effects on cell proliferation is the result of off-target effects. Nevertheless, it also revealed that certain splice variants of Sema4B are important for the ability of glioma cells to grow as individual clones.

Hyperglycemia Impairs Neutrophil Mobilization Leading to Enhanced Metastatic Seeding.

Preexisting diabetes is a risk factor for the development of multiple types of cancer. Additionally, diabetic patients face a poorer prognosis when diagnosed with cancer. To gain insight into the effects of hyperglycemia, a hallmark of diabetes, on tumor growth and metastatic progression, we combined mouse models of cancer and hyperglycemia. We show that while hyperglycemia attenuates primary tumor growth, it concomitantly increases metastatic seeding in a distant organ. We further show that the increase in metastatic seeding is due to impaired secretion of granulocyte colony-stimulating factor (G-CSF) and impaired neutrophil mobilization. Normalizing blood glucose levels using insulin rescues neutrophil recruitment and tumor growth and concomitantly reduces metastatic seeding. These results provide links among hyperglycemia-induced changes in neutrophil mobilization, primary tumor growth, and metastatic progression. Furthermore, our observations highlight the importance of normalizing blood glucose levels in hyperglycemic cancer patients.

miR-27a and miR-27a* contribute to metastatic properties of osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents and young adults. The essential mechanisms underlying osteosarcomagenesis and progression continue to be obscure. MicroRNAs (miRNAs) have far-reaching effects on the cellular biology of development and cancer. We recently reported that unique miRNA signatures associate with the pathogenesis and progression of OS. Of particular interest, we found that higher expression of miR-27a is associated with clinical metastatic disease. We report here that overexpression of miR-27a/miR-27a*, a microRNA pair derived from a single precursor, promotes pulmonary OS metastases formation. By contrast, sequestering miR-27a/miR-27a* by sponge technology suppressed OS cells invasion and metastases formation. miR-27a/miR-27a* directly repressed CBFA2T3 expression among other target genes. We demonstrated that CBFA2T3 is downregulated in majority of OS samples and its over expression significantly attenuated OS metastatic process mediated by miR-27a/miR-27a* underscoring CBFA2T3 functions as a tumor suppressor in OS. These findings establish that miR-27a/miR-27a* pair plays a significant role in OS metastasis and proposes it as a potential diagnostic and therapeutic target in managing OS metastases.

MiR-520d-5p directly targets TWIST1 and downregulates the metastamiR miR-10b.

MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.

Characterization of WWOX inactivation in murine mammary gland development.

The WW domain-containing oxidoreductase (WWOX) is commonly inactivated in multiple human cancers, including breast cancer. Wwox null mice die prematurely precluding adult tumor analysis. Nevertheless, aging Wwox-heterozygous mice at C3H genetic background develop higher incidence of mammary tumors. We recently generated a Wwox conditional knockout mouse in which loxp sites flank exon 1 in the Wwox allele and showed that total ablation of WWOX in these mice resembles that of conventional targeting of Wwox. Here, we report the characterization of WWOX ablation in mouse mammary gland using MMTV-Cre transgenic line. We demonstrated that WWOX ablation leads to impaired mammary ductal growth. Moreover, targeted deletion of WWOX is associated with increased levels of fibronectin, a component of the extracellular matrix. In addition, we showed that shRNA knockdown of WWOX in MCF10A breast epithelial cells dramatically increased fibronectin and is associated with enhanced cell survival and impaired growth in three-dimensional culture Matrigel assay. Taken together our results are consistent with a critical role for WWOX in normal breast development and tumorigenesis.