Bazedoxifene is a novel IL-6/GP130 inhibitor for treating triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) has been ranked as one of the devastating malignancy worldwide. Its disease progression and treatment obstacle is associated with the negligible expression of estrogen receptors (ER-), progesterone receptors (PR-), and HER2 (HER2-). Due to a lack of growth hormone receptors, TNBC is desperately demanding effective therapeutic regimens. A growing body of evidence indicated that glycoprotein 130 kDa (GP130), the pivotal mediator involved in interleukin 6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) signaling pathways, is strongly correlated with tumor progression. Therefore, GP130 could become a novel target for treating TNBC. In our earlier studies, we demonstrated bazedoxifene as being a novel GP130 inhibitor. METHODS: In the current report, anti-tumor effect of bazedoxifene on TNBC was further evaluated in TNBC cell lines SUM159, MDA-MB-231, and MDA-MB-468. We assessed anti-TNBC potency of bazedoxifene by carrying out various analysis encompassing western blot, cell proliferation, cell migration, colony formation, and growth of tumors in the xenograft mice. RESULTS: Our findings demonstrated that bazedoxifene not only decreased the expression of P-STAT3, IL-6/GP130-mediated downstream target genes P-AKT and P-ERK, but also blocked mitogen effects stimulated by IL-6, including cell viability, and overall cell survive, proliferation as well as cell migration. Likewise in laboratory animal model, tumor growth in mice was remarkably suppressed by bazedoxifene via an oral administration route. Combinational treatment of bazedoxifene plus the conventional chemotherapeutic agent, paclitaxel, synergistically impeded cell viability, colony formation, and cell migration far more significantly than the one from single-drug alone. CONCLUSIONS: Taken together, our data suggest that bazedoxifene may be developed as a promising small molecular therapeutic agent for eradicating TNBC intrinsically associated with constitutively active IL-6/GP130/STAT3 signaling cascade.

Jumping translocations, a novel finding in chronic lymphocytic leukaemia.

A jumping translocation (JT) is a rare cytogenetic aberration that can occur in haematological malignancy. It involves the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes, typically in different cells. In this study, we describe the first series of chronic lymphocytic leukaemia (CLL) patients with JTs reported to date. Following a review of 878 CLL patient karyotypes, we identified 26 patients (3%) with 97 JTs. The most commonly occurring breakpoint in these translocations was 17p11.2. Loss of TP53 was identified prior to or at the same time as JT in 23 of 26 patients (88%). All patients eventually developed a complex karyotype. All but one patient has required treatment for CLL, with estimated median time to treatment of 11·5 months. This study establishes JTs as a recurrent abnormality found in CLL patients with aggressive disease. JTs contribute to complex karyotypes and, in many cases, are involved in chromosomal rearrangements that result in loss of the tumour suppressor gene TP53.

Polarization of Cancer-Associated Macrophages Maneuver Neoplastic Attributes of Pancreatic Ductal Adenocarcinoma.

Mounting evidence links the phenomenon of enhanced recruitment of tumor-associated macrophages towards cancer bulks to neoplastic growth, invasion, metastasis, immune escape, matrix remodeling, and therapeutic resistance. In the context of cancer progression, naïve macrophages are polarized into M1 or M2 subtypes according to their differentiation status, gene signatures, and functional roles. While the former render proinflammatory and anticancer effects, the latter subpopulation elicits an opposite impact on pancreatic ductal adenocarcinoma. M2 macrophages have gained increasing attention as they are largely responsible for molding an immune-suppressive landscape. Through positive feedback circuits involving a paracrine manner, M2 macrophages can be amplified by and synergized with neighboring neoplastic cells, fibroblasts, endothelial cells, and non-cell autonomous constituents in the microenvironmental niche to promote an advanced disease state. This review delineates the molecular cues expanding M2 populations that subsequently convey notorious clinical outcomes. Future therapeutic regimens shall comprise protocols attempting to abolish environmental niches favoring M2 polarization; weaken cancer growth typically assisted by M2; promote the recruitment of tumoricidal CD8+ T lymphocytes and dendritic cells; and boost susceptibility towards gemcitabine as well as other chemotherapeutic agents.

Dichotomy effects of Akt signaling in breast cancer.

BACKGROUND: The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion. RESULTS: We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting. CONCLUSION: We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.

Breast Cancer Tumor Microenvironment and Molecular Aberrations Hijack Tumoricidal Immunity.

Breast cancer is the most common malignancy among females in western countries, where women have an overall lifetime risk of >10% for developing invasive breast carcinomas. It is not a single disease but is composed of distinct subtypes associated with different clinical outcomes and is highly heterogeneous in both the molecular and clinical aspects. Although tumor initiation is largely driven by acquired genetic alterations, recent data suggest microenvironment-mediated immune evasion may play an important role in neoplastic progression. Beyond surgical resection, radiation, and chemotherapy, additional therapeutic options include hormonal deactivation, targeted-signaling pathway treatment, DNA repair inhibition, and aberrant epigenetic reversion. Yet, the fatality rate of metastatic breast cancer remains unacceptably high, largely due to treatment resistance and metastases to brain, lung, or bone marrow where tumor bed penetration of therapeutic agents is limited. Recent studies indicate the development of immune-oncological therapy could potentially eradicate this devastating malignancy. Evidence suggests tumors express immunogenic neoantigens but the immunity towards these antigens is frequently muted. Established tumors exhibit immunological tolerance. This tolerance reflects a process of immune suppression elicited by the tumor, and it represents a critical obstacle towards successful antitumor immunotherapy. In general, immune evasive mechanisms adapted by breast cancer encompasses down-regulation of antigen presentations or recognition, lack of immune effector cells, obstruction of anti-tumor immune cell maturation, accumulation of immunosuppressive cells, production of inhibitory cytokines, chemokines or ligands/receptors, and up-regulation of immune checkpoint modulators. Together with altered metabolism and hypoxic conditions, they constitute a permissive tumor microenvironment. This article intends to discern representative incidents and to provide potential innovative therapeutic regimens to reinstate tumoricidal immunity.

Seed in soil, with an epigenetic view.

It is becoming increasingly evident that discrete genetic alterations in neoplastic cells alone cannot explain multistep carcinogenesis whereby tumor cells are able to express diverse phenotypes during the complex phases of tumor development and progression. The epigenetic model posits that the host microenvironment exerts an initial, inhibitory constraint on tumor growth that is followed by acceleration of tumor progression through complex cell-matrix interactions. This review emphasizes the epigenetic aspects of breast cancer development in light of such interactions between epithelial cells ("seed") and the tumor microenvironment ("soil"). Our recent research findings suggest that epigenetic perturbations induced by the tumor microenvironment may play a causal role in promoting breast cancer development. It is believed that abrogation of these initiators could offer a promising therapeutic strategy.

Promoter hypermethylation of FBXO32, a novel TGF-beta/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer.

Resistance to TGF-beta is frequently observed in ovarian cancer, and disrupted TGF-beta/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-beta/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-beta/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan-Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, P<0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-beta/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer.

Identification of candidate epigenetic biomarkers for ovarian cancer detection.

Ovarian cancer ranks the most lethal among gynecologic neoplasms in women. To develop potential biomarkers for diagnosis, we have identified five novel genes (CYP39A1, GTF2A1, FOXD4L4, EBP, and HAAO) that are hypermethylated in ovarian tumors, compared with the non-malignant normal ovarian surface epithelia, using the quantitative methylation-specific polymerase chain reactions. Interestingly enough, multivariate Cox regression analysis has identified hypermethylation of CYP39A1 correlated with an increase rate of relapsing (P=0.032, hazard ratio >1). Concordant hypermethylation in at least three loci was observed in 50 out of 55 (91%) of ovarian tumors examined. The test sensitivity and specificity were assessed to be 96 and 67% for CYP39A1; 95 and 88% for GTF2A1; 93 and 67% for FOXD4L4; 81 and 67% for EBP; 89 and 82% for HAAO, respectively. Our data have identified, for the first time, GTF2A1 alone, or GTF2A1 plus HAAO are excellent candidate biomarkers for detecting this disease. Moreover, the known functions of these gene products further implicate dysregulated transcriptional control, cholesterol metabolism, or synthesis of quinolinic acids, may play important roles in attributing to ovarian neoplasm. Molecular therapies, by reversing the aberrant epigenomes using inhibitory agents or by abrogating the upstream signaling pathways that convey the epigenomic perturbations, may be developed into promising treatment regimens.

Stress-associated hormone, norepinephrine, increases proliferation and IL-6 levels of human pancreatic duct epithelial cells and can be inhibited by the dietary agent, sulforaphane.

In current literature there is evidence that psychological factors can affect the incidence and progression of some cancers. Data obtained from animal models support the hypothesis that stress can be a cofactor. The underlying mechanisms for the association between psychological factors and pancreatic cancer are very poorly understood. In this study, we examined the possible growth promoting effects of the stress-associated hormone, norepinephrine, on immortalized human pancreatic duct epithelial cells. Our results suggest that norepinephrine can increase cell proliferation of human pancreatic duct epithelial cells. We also evaluated the ability of norepinephrine to induce interleukin-6 (IL-6), interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF). All of which may promote oncogenesis of immortalized human pancreatic duct epithelial cells. We found that norepinephrine can increase the IL-6 and VEGF but not IL-10 levels secreted by human pancreatic duct epithelial cells. Since norepinephrine can increase cell proliferation of human pancreatic duct epithelial cells, we performed further testing to see if dietary agents, sulforaphane and resveratrol, can inhibit norepinephrine-mediated increases in cell proliferation in human pancreatic duct epithelial cells. Interestingly, our results demonstrated that sulforaphane but not resveratrol inhibits norepinephrine-mediated increases in cell viability in human pancreatic duct epithelial cells. Furthermore, sulforaphane also inhibits norepinephrine-mediated increase of the IL-6 levels but not VEGF levels. Our study is the first to demonstrate that stress-associated hormone, norepinephrine, can increase the cell proliferation and IL-6 levels of human pancreatic duct epithelial cells, which can be inhibited by sulforaphane, a chemopreventive agent and a natural compound from the Cruciferous vegetables.

STAT3 can be activated through paracrine signaling in breast epithelial cells.

BACKGROUND: Many cancers, including breast cancer, have been identified with increased levels of phosphorylated or the active form of Signal Transducers and Activators of Transcription 3 (STAT3) protein. However, whether the tumor microenvironment plays a role in this activation is still poorly understood. METHODS: Conditioned media, which contains soluble factors from MDA-MB-231 and MDA-MB-468 breast cancer cells and breast cancer associated fibroblasts, was added to MCF-10A breast epithelial and MDA-MB-453 breast cancer cells. The stimulation of phosphorylated STAT3 (p-STAT3) levels by conditioned media was assayed by Western blot in the presence or absence of neutralized IL-6 antibody, or a JAK/STAT3 inhibitor, JSI-124. The stimulation of cell proliferation in MCF-10A cells by conditioned media in the presence or absence of JSI-124 was subjected to MTT analysis. IL-6, IL-10, and VEGF levels were determined by ELISA analysis. RESULTS: Our results demonstrated that conditioned media from cell lines with constitutively active STAT3 are sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels. This signaling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and is persistent for at least 24 hours. ELISA analysis confirmed a correlation between elevated levels of IL-6 production and p-STAT3. Neutralization of the IL-6 ligand or gp130 was sufficient to block increased levels of p-STAT3 (Y705) in treated cells. Furthermore, soluble factors within the MDA-MB-231 conditioned media were also sufficient to stimulate an increase in IL-6 production from MCF-10A cells. CONCLUSION: These results demonstrate STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signaling through soluble factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The induction of STAT3 phosphorylation is through the IL-6/JAK pathway and appears to be associated with cell proliferation. Understanding how IL-6 and other soluble factors may lead to STAT3 activation via the tumor microenvironment will provide new therapeutic regimens for breast carcinomas and other cancers with elevated p-STAT3 levels.

Inhibition of interleukin 8/C­X-C chemokine receptor 1,/2 signaling reduces malignant features in human pancreatic cancer cells.

Interactions between interleukin (IL)-8 and its receptors, C­X-C chemokine receptor 1, (CXCR1) and CXCR2 serve crucial roles in increasing cancer progression. Inhibition of this signaling pathway has yielded promising results in a number of human cancers, including breast, melanoma and colon. However, the effects of CXCR1/2 antagonist treatment on pancreatic cancer remain unclear. The present study aimed to demonstrate that treatment with the clinical grade CXCR1/2 antagonist, reparixin, or the newly discovered CXCR1/2 antagonist, SCH527123, may result in a reduction of the malignant features associated with this lethal cancer. The effects of reparixin or SCH527123 exposure on human pancreatic cancer cell lines BxPC­3, HPAC, Capan­1, MIA PaCa­2, and AsPC­1 were examined in regard to cell proliferation, cell viability, colony formation and migration. The effects of CXCR1/2 inhibition on the protein expression of well-known downstream effectors, including phosphorylated (p)-signal transducer and activator of transcription 3 (STAT3), p­RAC­α serine/threonine-protein kinase (p­AKT), p­extracellular signal-regulated kinase (p­ERK1/2) and p­ribosomal protein S6 (p­S6), were assessed by western blotting assays. The effects of IL­8 signaling on the proliferative activities intrinsic to the human pancreatic cancer cell lines Capan­1, AsPC­1 and HPAC were examined by bromodeoxyuridine assay. Treatment with either reparixin or SCH527123 yielded dose-dependent growth suppressive effects on HPAC, Capan­1 and AsPC­1 cells that may have otherwise undergone robust proliferation upon IL­8 stimulation. In addition, reparixin or SCH527123 treatment inhibited CXCR1/2-mediated signal transduction, as demonstrated by the decreased phosphorylation levels of effector molecules STAT3, AKT, ERK and S6 that are downstream of the IL­8/CXCR1/2 signaling cascade in HPAC cells. These data were in close agreement with the reduced cell migration and colony formation. Results from the present study suggested that reparixin and SCH527123 may be promising therapeutic agents for the treatment of pancreatic cancer by inhibiting the IL­8/CXCR1/2 signaling cascade.

Seed-in-Soil: Pancreatic Cancer Influenced by Tumor Microenvironment.

Pancreatic ductal adenocarcinoma is a fatal malignancy with a five-year survival rate lower than 7%, and most patients dying within six months of diagnosis. The factors that contribute to the aggressiveness of the disease include, but are not limited to: late diagnosis, prompt metastasis to adjacent vital organs, poor response, and resistance to anticancer treatments. This malignancy is uniquely associated with desmoplastic stroma that accounts for 80% of tumor mass. Understanding the biology of stroma can aid the discovery of innovative strategies for eradicating this lethal cancer in the future. This review highlights the critical components in the stroma and how they interact with the cancer cells to convey the devastating tumor progression.

Modulation of signal transducer and activator of transcription 3 activities by p53 tumor suppressor in breast cancer cells.

The constitutive activation of the Stat3 oncogene product and mutation of the p53 tumor suppressor are both frequently detected in human breast cancer. We sought to determine whether there is functional regulation of Stat3 by wild-type (wt) p53. We demonstrate that expression of wt p53, but not mutant p53, significantly diminished phosphorylation of Stat3, reduced Stat3 DNA binding activity, and inhibited Stat3-dependent transcriptional activity in breast cancer cells expressing constitutively active Stat3. Expression of wt p53 did not cause a reduction in the phosphorylation of three unrelated protein kinases in other signal transduction pathways, AKT, extracellular signal-regulated kinase (ERK)1, and ERK2 or a reduction of phosphorylation of epidermal growth factor receptor. Furthermore, the expression of the p53 downstream target, p21(WAF-1), did not have an inhibitory effect on Stat3 phosphorylation. Wt p53 also induced significant apoptosis in breast cancer cell lines that express constitutively active Stat3. Interestingly, the p53-dependent apoptosis occurred in the presence of high levels of phosphorylated AKT and ERK1/2. Therefore, these findings demonstrate a novel p53-dependent cellular process that regulates Stat3 phosphorylation and activity.

Feedback Activation of STAT3 as a Cancer Drug-Resistance Mechanism.

Signal transducer and activator of transcription 3 (STAT3) plays crucial roles in several cellular processes such as cell proliferation and survival, and has been found to be aberrantly activated in many cancers. Much research has explored the leading mechanisms for regulating the STAT3 pathway and its role in promoting tumorigenesis. We focus here on recent evidence suggesting that feedback activation of STAT3 plays a prominent role in mediating drug resistance to a broad spectrum of targeted cancer therapies and chemotherapies. We highlight the potential of co-targeting STAT3 and its primary target to overcome drug resistance, and provide perspective on repurposing clinically approved drugs as STAT3 pathway inhibitors, in combination with the FDA-approved receptor tyrosine kinase (RTK) inhibitors, to improve clinical outcome of cancer treatment.

Evaluation of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24- subpopulations of breast cancer cells.

BACKGROUND: STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH(+)), or cell surface molecule CD44-positive (CD44(+)) but CD24-negative (CD24(-)) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells is unknown. METHODS AND RESULTS: We examined STAT3 activation in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH(+)) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH(-)) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH(+) subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH(+) subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH(+) subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH(+) breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH(+) cells were further selected for the stem cell markers CD44(+) and CD24(-). CONCLUSION: These studies demonstrate an important role for STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

Epigenetic repression of RARRES1 is mediated by methylation of a proximal promoter and a loss of CTCF binding.

BACKGROUND: The cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We first compared methylation occurring at the sequences (-664~+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (-664~-86), while the downstream element (-85~+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. CONCLUSION/SIGNIFICANCE: This report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.

STAT3 is necessary for proliferation and survival in colon cancer-initiating cells.

STAT3 is constitutively activated in colon cancer but its contributions in cancer-initiating cells have not been explored. In this study, we characterized STAT3 in aldehyde dehydrogenase (ALDH)-positive (ALDH(+)) and CD133-positive (CD133(+)) subpopulations of human colon tumor cells that exhibited more potent tumor-initiating ability than ALDH(-)/CD133(-) cells in tumor xenograft assays in mice. We found that ALDH(+)/CD133(+) cells expressed higher levels of the active phosphorylated form of STAT3 than either ALDH(-)/CD133(-) or unfractionated colon cancer cells. STAT3 inhibition by RNA interference-mediated knockdown or small-molecule inhibitors LLL12 or Stattic blocked downstream target gene expression, cell viability, and tumorsphere-forming capacity in cancer-initiating cells. Similarly, treatment of mouse tumor xenografts with STAT3 short hairpin RNA (shRNA), interleukin 6 shRNA, or LLL12 inhibited tumor growth. Our results establish that STAT3 is constitutively activated in colon cancer-initiating cells and that these cells are sensitive to STAT3 inhibition. These findings establish a powerful rationale to develop STAT3 inhibitory strategies for treating advanced colorectal cancers.

Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.

Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.