ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer.

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR.

Can supplementation of phytoestrogens/insoluble fibers help the management of duodenal polyps in familial adenomatous polyposis?

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disorder, and prophylactic colectomy has been shown to decrease the incidence of colorectal cancer (CRC). Duodenal cancer and desmoids are now the leading causes of death in FAP. We evaluate whether 3 months of oral supplementation with a patented blend of phytoestrogens and indigestible insoluble fibers (ADI) help the management of FAP patients with ileal pouch-anal anastomosis (IPAA). In a prospective open label study, we enrolled 15 FAP patients with IPAA and duodenal polyps who underwent upper gastrointestinal endoscopy at baseline and after 3 months of treatment. The primary endpoint was the change in gene expression in polyp mucosa, whereas the secondary endpoint was the reduction in polyp number and size. After 3 months of ADI treatment, all patients showed a reduction in the number and size of duodenal polyps (P = 0.021). Analysis of the expression of CRC promoting/inhibiting genes in duodenal polyps biopsies demonstrated that different CRC-promoting genes (PCNA, MUC1 and COX-2) were significantly downregulated, whereas CRC-inhibiting genes (ER-ß and MUC2) were significantly upregulated after ADI treatment. In conclusion, ADI proved to be safe and effective, and its long-term effects on FAP patients need further investigation. Judging from the results we observed on COX-2 and miR-101 expression, the short-term effects of ADI treatment could be comparable with those obtained using COX-2 inhibitors, with the advantage of being much more tolerable in chronic therapies and void of adverse events.

Dietary Geraniol by Oral or Enema Administration Strongly Reduces Dysbiosis and Systemic Inflammation in Dextran Sulfate Sodium-Treated Mice.

(Trans)-3,7-Dimethyl-2,6-octadien-1-ol, commonly called geraniol (Ge-OH), is an acyclic monoterpene alcohol with well-known anti-inflammatory, antitumoral, and antimicrobial properties. It is widely used as a preservative in the food industry and as an antimicrobial agent in animal farming. The present study investigated the role of Ge-OH as an anti-inflammatory and anti-dysbiotic agent in the dextran sulfate sodium (DSS)-induced colitis mouse model. Ge-OH was orally administered to C57BL/6 mice at daily doses of 30 and 120 mg kg((-1)) body weight, starting 6 days before DSS treatment and ending the day after DSS removal. Furthermore, Ge-OH 120 mg kg((-1)) dose body weight was administered via enema during the acute phase of colitis to facilitate its on-site action. The results show that orally or enema-administered Ge-OH is a powerful antimicrobial agent able to prevent colitis-associated dysbiosis and decrease the inflammatory systemic profile of colitic mice. As a whole, Ge-OH strongly improved the clinical signs of colitis and significantly reduced cyclooxygenase-2 (COX-2) expression in colonocytes and in the gut wall. Ge-OH could be a powerful drug for the treatment of intestinal inflammation and dysbiosis.

MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells. RESULTS: Herein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells. CONCLUSIONS: Altogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.

Cyclooxygenase-2 silencing for the treatment of colitis: a combined in vivo strategy based on RNA interference and engineered Escherichia coli.

Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColi(shCOX2)), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColi(shCOX2) in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColi(shCOX2)-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColi(shCOX2) strategy as a promising tool for molecular therapy in intestinal inflammatory diseases.

Longitudinal analysis of inflammation and microbiota dynamics in a model of mild chronic dextran sulfate sodium-induced colitis in mice.

AIM: To characterize longitudinally the inflammation and the gut microbiota dynamics in a mouse model of dextran sulfate sodium (DSS)-induced colitis. METHODS: In animal models, the most common method used to trigger colitis is based on the oral administration of the sulfated polysaccharides DSS. The murine DSS colitis model has been widely adopted to induce severe acute, chronic or semi-chronic colitis, and has been validated as an important model for the translation of mice data to human inflammatory bowel disease (IBD). However, it is now clear that models characterized by mild intestinal damage are more accurate for studying the effects of therapeutic agents. For this reason, we have developed a murine model of mild colitis to study longitudinally the inflammation and microbiota dynamics during the intestinal repair processes, and to obtain data suitable to support the recovery of gut microbiota-host homeostasis. RESULTS: All plasma cytokines evaluated, except IL-17, began to increase (P < 0.05), after 7 d of DSS administration. IL-17 only began to increase 4 d after DSS withdrawal. IL-1ß and IL-17 continue to increase during the recovery phase, even when clinical signs of colitis had disappeared. IL-6, IL-10 and IFN-γ reached their maxima 4 d after DSS withdrawal and decreased during the late recovery phase. TNFα reached a peak (a three- fold increase, P < 0.05), after which it slightly decreased, only to increase again close to the end of the recovery phase. DSS administration induced profound and rapid changes in the mice gut microbiota. After 3 d of DSS administration, we observed a major reduction in Bacteroidetes/Prevotella and a corresponding increase in Bacillaceae, with respect to control mice. In particular, Bacteroidetes/Prevotella decreased from a relative abundance of 59.42%-33.05%, while Bacillaceae showed a concomitant increase from 2.77% to 10.52%. Gut microbiota rapidly shifted toward a healthy profile during the recovery phase and returned normal 4 d after DSS withdrawal. Cyclooxygenase 2 expression started to increase 4 d after DSS withdrawal (P < 0.05), when dysbiosis had recovered, and continued to increase during the recovery phase. Taken together, these data indicated that a chronic phase of intestinal inflammation, characterized by the absence of dysbiosis, could be obtained in mice using a single DSS cycle. CONCLUSION: Dysbiosis contributes to the local and systemic inflammation that occurs in the DSS model of colitis; however, chronic bowel inflammation is maintained even after recovery from dysbiosis.

Loss of miR-101 expression promotes Wnt/ß-catenin signalling pathway activation and malignancy in colon cancer cells.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR-101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR-101 re-expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR-101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR-101 expression and important CRC pro-malignant features, such as inflammation, activation of the Wnt/ß-catenin signalling pathway and epithelial-mesenchymal transition (EMT). We then propose that up-regulated miR-101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR-101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis.

Drug delivery by polymeric micelles: an in vitro and in vivo study to deliver lipophilic substances to colonocytes and selectively target inflamed colon.

Colitis is the term used for chronic inflammatory bowel diseases at substantially increased risk of developing a form of colorectal cancer (CRC) known as colitis-associated cancer. In our study we synthesized core-shell polymeric micelles obtained by self-assembly of block copolymers for high efficiency delivery of anti-inflammatory and anti-cancer compounds to colonocytes and colon mucosa. We achieved an efficient intracellular delivery of these hydrophobic compounds (prednisone, retinoic acid and doxorubicin) to cultured colonocytes without cellular toxicity. The efficacy of retinoic acid and doxorubicin administration was significantly increased using these nanosized carriers. Moreover, these polymeric micelles have been shown to overcome the multidrug resistance efflux mechanism effectively delivering doxorubicin to multidrug-resistant colon cancer cells. These nanocarriers are also suitable for selective in vivo delivery of lipophilic drugs by enema administration to the inflamed colon tissue, specifically targeting the inflamed mucosa. FROM THE CLINICAL EDITOR: This team of investigators studied polymeric micelles as highly efficient drug delivery systems enabling intracellular delivery of hydrophobic compounds (prednisone, retinoic acid, and doxorubicin) to cultured colonocytes without cellular toxicity, also demonstrating beneficial in vivo effects.