The epithelial-mesenchymal transition generates cells with properties of stem cells.

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Design of BET Inhibitor Bottlebrush Prodrugs with Superior Efficacy and Devoid of Systemic Toxicities.

Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic indexes (TIs) of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive synthesis and optimization. Here, using bromodomain and extra-terminal (BET) protein inhibitors (BETi)-a class of epigenetic regulators with proven anticancer potential but clinical development hindered in large part by narrow TIs-we introduce a macromolecular prodrug platform that overcomes these challenges. Through tuning of traceless linkers appended to a "bottlebrush prodrug" scaffold, we demonstrate correlation of in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, enabling the predictive design of novel BETi prodrugs with enhanced antitumor efficacies and devoid of dose-limiting toxicities in a syngeneic triple-negative breast cancer murine model. This work may have immediate clinical implications, introducing a platform for predictive prodrug design and potentially overcoming hurdles in drug development.

Molecular definition of breast tumor heterogeneity.

Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells purified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations.

Altered antisense-to-sense transcript ratios in breast cancer.

Transcriptome profiling studies suggest that a large fraction of the genome is transcribed and many transcripts function independent of their protein coding potential. The relevance of noncoding RNAs (ncRNAs) in normal physiological processes and in tumorigenesis is increasingly recognized. Here, we describe consistent and significant differences in the distribution of sense and antisense transcripts between normal and neoplastic breast tissues. Many of the differentially expressed antisense transcripts likely represent long ncRNAs. A subset of genes that mainly generate antisense transcripts in normal but not cancer cells is involved in essential metabolic processes. These findings suggest fundamental differences in global RNA regulation between normal and cancer cells that might play a role in tumorigenesis.

Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality.

BACKGROUND: We have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features. RESULTS: Here we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue. CONCLUSIONS: In summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.

Gene expression profiling of human breast tissue samples using SAGE-Seq.

We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein-coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease.

Activation of RalA is critical for Ras-induced tumorigenesis of human cells.

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

Cell type-specific DNA methylation patterns in the human breast.

Cellular identity and differentiation are determined by epigenetic programs. The characteristics of these programs in normal human mammary epithelium and their similarity to those in stem cells are unknown. To begin investigating these issues, we analyzed the DNA methylation and gene expression profiles of distinct subpopulations of mammary epithelial cells by using MSDK (methylation-specific digital karyotyping) and SAGE (serial analysis of gene expression). We identified discrete cell-type and differentiation state-specific DNA methylation and gene expression patterns that were maintained in a subset of breast carcinomas and correlated with clinically relevant tumor subtypes. CD44+ cells were the most hypomethylated and highly expressed several transcription factors with known stem cell function including HOXA10 and TCF3. Many of these genes were also hypomethylated in BMP4-treated compared with undifferentiated human embryonic stem (ES) cells that we analyzed by MSDK for comparison. Further highlighting the similarity of epigenetic programs of embryonic and mammary epithelial cells, genes highly expressed in CD44+ relative to more differentiated CD24+ cells were significantly enriched for Suz12 targets in ES cells. The expression of FOXC1, one of the transcription factors hypomethylated and highly expressed in CD44+ cells, induced a progenitor-like phenotype in differentiated mammary epithelial cells. These data suggest that epigenetically controlled transcription factors play a key role in regulating mammary epithelial cell phenotypes and imply similarities among epigenetic programs that define progenitor cell characteristics.

Identification of CD44v6(+)/CD24- breast carcinoma cells in primary human tumors by quantum dot-conjugated antibodies.

Breast carcinoma cells with the CD44+/CD24(low) phenotype have been reported to exhibit 'cancer stem cell' (CSC) characteristics on the basis of their enhanced tumorigenicity and self-renewal potential in immunodeficient mice. We used immunohistochemistry to study the expression of these proteins in whole tissue sections of human breast carcinoma. We found that the fraction of CD44v6+ cells is higher in estrogen receptor-positive carcinomas after neoadjuvant chemotherapy. We also performed double immunohistochemistry for CD44v6 and for the proliferation marker Ki67. We found that the relative number of quiescent carcinoma cells is higher in the CD44v6+ population than in the CD44v6- population in specific carcinoma subtypes. We then used quantum dots and spectral imaging to increase the number of antigens that could be visualized in a single tissue section. We found that anti-CD44v6 and CD24 antibodies that were directly conjugated to quantum dots retained their ability to recognize antigen in formalin-fixed, paraffin-embedded tissue sections. We then performed triple staining for CD44v6, CD24 and Ki67 to assess the proliferation of each sub-population of breast carcinoma cells. Our results identify differences between CD44v6-positive and CD44v6-negative breast carcinoma cells in vivo and provide a proof of principle that quantum dot-conjugated antibodies can be used to study specific sub-populations of cancer cells defined by multiple markers in a single tissue section.

The cancer stem cell hypothesis: in search of definitions, markers, and relevance.

Cancer is a disease of genes. Inherited or somatic alterations in genes are what make a normal cell ignore growth-controlling signals and form a tumor that eventually leads to the destruction of the organism. Based on accumulated knowledge on the genetic composition of cancer cells, the clonal evolution model of tumorigenesis was established, which explains multiple aspects of human disease and clinical observations. However, the recently popularized cancer stem cell hypothesis questions that all or most tumor cells can participate in tumor evolution and restricts this property to a subset of them defined as 'cancer stem cells' due to their stem cell-like characteristics. Enthusiasm surrounding this area of investigation and its presumed clinical implications led to a spurt of studies in various cancer types and model systems. Rigorous study design and critical data interpretation have to be employed to test the scientific and clinical relevance of the cancer stem cell hypothesis and its relationship to the clonal evolution model.

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.

Publisher Correction: Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'

RalA but not RalB enhances polarized delivery of membrane proteins to the basolateral surface of epithelial cells.

RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.

Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles.

The Ral proteins are members of the Ras superfamily of GTPases. Because they reside in synaptic vesicles, we used transgenic mice expressing a dominant inhibitory form of Ral to investigate the role of Ral in neurosecretion. Using a synaptosomal secretion assay, we found that while K(+)-evoked secretion of glutamate was normal, protein kinase C-mediated enhancement of glutamate secretion was suppressed in the mutant mice. Since protein kinase C effects on secretion have been shown to be due to enhancement of the size of the readily releasable pool of synaptic vesicles docked at the plasma membrane, we directly measured the refilling of this readily releasable pool of synaptic vesicles after Ca(2+)-triggered exocytosis. Refilling of the readily releasable pool was suppressed in synaptosomes from mice expressing dominant inhibitory Ral. Moreover, we found that protein kinase C and calcium-induced phosphorylation of proteins thought to influence synaptic vesicle function, such as MARCKS, synapsin, and SNAP-25, were all reduced in synaptosomes from these transgenic mice. Concomitant with these studies, we searched for new functions of Ral by detecting proteins that specifically bind to it in cells. Consistent with the phenotype of the transgenic mice described above, we found that active but not inactive RalA binds to the Sec6/8 (exocyst) complex, whose yeast counterpart is essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. These findings demonstrate a role for Ral-GTPase signaling in the modulation of the readily releasable pool of synaptic vesicles and suggest the possible involvement of Ral-Sec6/8 (exocyst) binding in modulation of synaptic strength.

Development and clinical validation of an in situ biopsy-based multimarker assay for risk stratification in prostate cancer.

PURPOSE: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. EXPERIMENTAL DESIGN: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish "favorable" versus "nonfavorable" pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). RESULTS: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on "false-negative" and "false-positive" rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non-GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). CONCLUSIONS: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy.

The JAK2/STAT3 signaling pathway is required for growth of CD44⁺CD24⁻ stem cell-like breast cancer cells in human tumors.

Intratumor heterogeneity is a major clinical problem because tumor cell subtypes display variable sensitivity to therapeutics and may play different roles in progression. We previously characterized 2 cell populations in human breast tumors with distinct properties: CD44+CD24- cells that have stem cell-like characteristics, and CD44-CD24+ cells that resemble more differentiated breast cancer cells. Here we identified 15 genes required for cell growth or proliferation in CD44+CD24- human breast cancer cells in a large-scale loss-of-function screen and found that inhibition of several of these (IL6, PTGIS, HAS1, CXCL3, and PFKFB3) reduced Stat3 activation. We found that the IL-6/JAK2/Stat3 pathway was preferentially active in CD44+CD24- breast cancer cells compared with other tumor cell types, and inhibition of JAK2 decreased their number and blocked growth of xenografts. Our results highlight the differences between distinct breast cancer cell types and identify targets such as JAK2 and Stat3 that may lead to more specific and effective breast cancer therapies.

Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer.

PURPOSE: To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage. EXPERIMENTAL DESIGN: We performed immunohistochemical analyses of 12 proteins [CD44, CD24, ALDH1, vimentin, osteonectin, EPCR, caveolin 1, connexin 43, cytokeratin 18 (CK18), MUC1, claudin 7, and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only, 135 cases of IDC with ductal carcinoma in situ (DCIS), 35 cases of DCIS with microinvasion, and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry. RESULTS: CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors, especially in luminal A subtype. The expression of vimentin, osteonectin, connexin 43, ALDH1, CK18, GATA3, and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells. CONCLUSIONS: Our findings suggest that in breast cancer, the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage.

Epigenetic patterns of embryonic and adult stem cells.

Embryonic stem (ES) cells are pluripotent cells that differentiate into all cell types of the organism. In adult, multipotent tissue-specific stem cells undergo multi-lineage differentiation to preserve normal tissue homeostasis and repair potential injuries. The maintenance of stem cells and their differentiation follows defined epigenetic programs, including DNA methylation, histone modifications and small non-coding RNAs that result in gene expression, morphologic and functional changes. Recently, we reported for the first time the comprehensive characterization of the in vivo gene expression and DNA methylation profiles of four distinct populations of normal human mammary epithelial cells and the identification of cell type-specific DNA methylation patterns with clinical relevance. Our results together with other studies suggest an important role for epigenetic regulation in stem cell self-renewal, pluripotency and differentiation, and imply that abnormalities in these processes may play a role in tumor initiation and progression.

No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas.

There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs.

The genomic landscapes of human breast and colorectal cancers.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene "mountains" and a much larger number of gene "hills" that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.