Kruppel-like factor 4 signals through microRNA-206 to promote tumor initiation and cell survival.

Tumor cell heterogeneity poses a major hurdle in the treatment of cancer. Mammary cancer stem-like cells (MaCSCs), or tumor-initiating cells, are highly tumorigenic sub-populations that have the potential to self-renew and to differentiate. These cells are clinically important, as they display therapeutic resistance and may contribute to treatment failure and recurrence, but the signaling axes relevant to the tumorigenic phenotype are poorly defined. The zinc-finger transcription factor Kruppel-like factor 4 (KLF4) is a pluripotency mediator that is enriched in MaCSCs. KLF4 promotes RAS-extracellular signal-regulated kinase pathway activity and tumor cell survival in triple-negative breast cancer (TNBC) cells. In this study, we found that both KLF4 and a downstream effector, microRNA-206 (miR-206), are selectively enriched in the MaCSC fractions of cultured human TNBC cell lines, as well as in the aldehyde dehydrogenase-high MaCSC sub-population of cells derived from xenografted human mammary carcinomas. The suppression of endogenous KLF4 or miR-206 activities abrogated cell survival and in vivo tumor initiation, despite having only subtle effects on MaCSC abundance. Using a combinatorial approach that included in silico as well as loss- and gain-of-function in vitro assays, we identified miR-206-mediated repression of the pro-apoptotic molecules programmed cell death 4 (PDCD4) and connexin 43 (CX43/GJA1). Depletion of either of these two miR-206-regulated transcripts promoted resistance to anoikis, a prominent feature of CSCs, but did not consistently alter MaCSC abundance. Consistent with increased levels of miR-206 in MaCSCs, the expression of both PDCD4 and CX43 was suppressed in these cells relative to control cells. These results identify miR-206 as an effector of KLF4-mediated prosurvival signaling in MaCSCs through repression of PDCD4 and CX43. Consequently, our study suggests that a pluripotency factor exerts prosurvival signaling in MaCSCs, and that antagonism of KLF4-miR-206 signaling may selectively target the MaCSC niche in TNBC.

Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer.

The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

Gli1 acts through Snail and E-cadherin to promote nuclear signaling by beta-catenin.

The Hedgehog pathway transcription factor Gli1 induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin (E-cad). E-cad is normally complexed with beta-catenin at the cell membrane. Loss of E-cad during developmental epithelial-mesenchymal transitions can switch beta-catenin from its role at adherens junctions to its role in nuclear transcription. During tumorigenesis it is unclear which pathways trigger this switch. In the current study, gain- and loss-of-function approaches identified E-cad as a selective inhibitor of transformation by Gli1, and Snail knockdown was rescued by downregulation of E-cad. Gli1 induced relocalization of beta-catenin from the cell membrane to the nucleus. The ability of wild-type or mutant alleles of E-cad to modulate transformation by Gli1 correlated with their ability to regulate localization of beta-catenin. Inhibition of Wnt-beta-catenin signaling by dominant negative Tcf4 selectively blocked in vitro transformation by Gli1. In Gli1-transgenic mice, infiltrating skin tumor cells expressed active, unphosphorylated beta-catenin. Our studies identify E-cad as a selective suppressor of transformation by Gli1 and point to the Sonic Hedgehog-Gli1 pathway as a key regulator of the beta-catenin switch in epithelial cells and cancers.

Snail induction is an early response to Gli1 that determines the efficiency of epithelial transformation.

Gli family members mediate constitutive Hedgehog signaling in the common skin cancer, basal cell carcinoma (BCC). Snail/Snai1 is rapidly induced by Gli1 in vitro, and is coexpressed with Gli1 in human hair follicles and skin tumors. In the current study, we generated a dominant-negative allele of Snail, SnaZFD, composed of the zinc-finger domain and flanking sequence. In promoter-reporter assays, SnaZFD blocked the activity of wild-type Snail on the E-cadherin promoter. Snail loss-of-function mediated by SnaZFD or by one of several short hairpin RNAs inhibited transformation of RK3E epithelial cells by Gli1. Conversely, enforced expression of Snail promoted transformation in vitro by Gli1, but not by other genes that were tested, including Notch1, ErbB2, and N-Ras. As observed for Gli1, wild-type Snail repressed E-cadherin in RK3E cells and induced blebbing of the cytoplasmic membrane. Induction of a conditional Gli1 transgene in the basal keratinocytes of mouse skin led to rapid upregulation of Snail transcripts and to cell proliferation in the interfollicular epidermis. Established Gli1-induced skin lesions exhibited molecular similarities to BCC, including loss of E-cadherin. The results identify Snail as a Gli1-inducible effector of transformation in vitro, and an early Gli1-responsive gene in the skin.

Increase of GKLF messenger RNA and protein expression during progression of breast cancer.

Genetic alterations found in carcinomas can alter specific regulatory pathways and provide a selective growth advantage by activation of transforming oncogenes. A subset of these genes, including wild-type alleles of GLI or c-MYC, and activated alleles of RAS or beta-catenin, exhibit transforming activity when expressed in diploid epithelial RK3E cells in vitro. By in vitro transformation of these cells, the zinc finger protein GKLF/KLF-4 was recently identified as a novel oncogene. Although GKLF is normally expressed in superficial, differentiating epithelial cells of the skin, oral mucosa, and gut, expression is consistently up-regulated in dysplastic epithelium and in squamous cell carcinoma of the oral cavity. In the current study, we used in situ hybridization, Northern blot analysis, and immunohistochemistry to detect GKLF at various stages of tumor progression in the breast, prostate, and colon. Overall, expression of GKLF mRNA was detected by in situ hybridization in 21 of 31 cases (68%) of carcinoma of the breast. Low-level expression of GKLF mRNA was observed in morphologically normal (uninvolved) breast epithelium adjacent to tumor cells. Increased expression was observed in neoplastic cells compared with adjacent uninvolved epithelium for 14 of 19 cases examined (74%). Ductal carcinoma in situ exhibited similar expression as invasive carcinoma, suggesting that GKLF is activated prior to invasion through the basement membrane. Expression as determined by Northern blot was increased in most breast tumor cell lines and in immortalized human mammary epithelial cells when these were compared with finite-life span human mammary epithelial cells. Alteration of GKLF expression was confirmed by the use of a novel monoclonal antibody that detected the protein in normal and neoplastic tissues in a distribution consistent with localization of the mRNA. In contrast to most breast tumors, expression of GKLF in tumor cells of colorectal or prostatic carcinomas was reduced or unaltered compared with normal epithelium. The results demonstrate that GKLF expression in epithelial compartments is altered in a tissue-type specific fashion during tumor progression, and suggest that increased expression of GKLF mRNA and protein may contribute to the malignant phenotype of breast tumors.

Alternatively spliced hBRF variants function at different RNA polymerase III promoters.

In yeast, a single form of TFIIIB is required for transcription of all RNA polymerase III (pol III) genes. It consists of three subunits: the TATA box-binding protein (TBP), a TFIIB-related factor, BRF, and B". Human TFIIIB is not as well defined and human pol III promoters differ in their requirements for this activity. A human homolog of yeast BRF was shown to be required for transcription at the gene-internal 5S and VA1 promoters. Whether or not it was also involved in transcription from the gene-external human U6 promoter was unclear. We have isolated cDNAs encoding alternatively spliced forms of human BRF that can complex with TBP. Using immunopurified complexes containing the cloned hBRFs, we show that while hBRF1 functions at the 5S, VA1, 7SL and EBER2 promoters, a different variant, hBRF2, is required at the human U6 promoter. Thus, pol III utilizes different TFIIIB complexes at structurally distinct promoters.

The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma.

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.

Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts.

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.

The zinc finger protein GLI induces cellular sensitivity to the mTOR inhibitor rapamycin.

The protein synthetic machinery is activated by diverse genetic alterations during tumor progression in vivo and represents an attractive target for cancer therapy. We show that rapamycin inhibits the induction of transformed foci in vitro by GLI, a transcription factor that functions in the sonic hedgehog-patched pathway in tumors. In control cells, which were nontransformed epithelioid RK3E cells and derivative c-MYC- or RAS-transformed sister cell lines, rapamycin inhibits mTOR and mTOR-dependent activities but increases global protein synthesis, perhaps by activating a feedback mechanism. In GLI-transformed cells, rapamycin inhibits global protein synthesis and turnover and prevents cellular proliferation. In contrast to its effects on protein synthesis, rapamycin affects bromodeoxyuridine incorporation and cell cycle occupancy of GLI cells and control cells to a similar extent. Rare, variant GLI cells isolated by selection in rapamycin are also drug-resistant for protein metabolism and for cell cycle progression through G1. Our results indicate that sensitivity to rapamycin can be induced by a specific oncogene and that inhibition of global protein metabolism is linked to the rapamycin-sensitive phenotype.

Oncogene expression cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF.

The function of several known oncogenes is restricted to specific host cells in vitro, suggesting that new genes may be identified by using alternate hosts. RK3E cells exhibit characteristics of epithelia and are susceptible to transformation by the G protein RAS and the zinc finger protein GLI. Expression cloning identified the major transforming activities in squamous cell carcinoma cell lines as c-MYC and the zinc finger protein gut-enriched Kruppel-like factor (GKLF)/epithelial zinc finger. In oral squamous epithelium, GKLF expression was detected in the upper, differentiating cell layers. In dysplastic epithelium, expression was prominently increased and was detected diffusely throughout the entire epithelium, indicating that GKLF is misexpressed in the basal compartment early during tumor progression. The results demonstrate transformation of epithelioid cells to be a sensitive and specific assay for oncogenes activated during tumorigenesis in vivo, and identify GKLF as an oncogene that may function as a regulator of proliferation or differentiation in epithelia.

Allelotype of head and neck squamous cell carcinoma.

To gain a better understanding of the molecular changes in head and neck squamous cell carcinoma, we tested every autosomal arm of 29 primary head and neck tumors for allelic loss. Fifty-eight microsatellite markers were used with at least two-thirds of patients informative for each chromosomal arm tested. A high frequency of allelic loss was found on chromosome 9p where 21 of 29 (72%) tumors had loss of heterozygosity for at least one polymorphic marker on this arm. Chromosomes 3, 11q, 13q, and 17p exhibited loss in over 50% of all informative cases, while chromosomes 4, 6p, 8, 14q, and 19q displayed loss in greater than 35% of all cases tested. Additionally, several other chromosomal arms exhibited loss of heterozygosity in 20 to 30% of tumors tested. This high frequency of allelic loss in these advanced stage neoplasms suggests multiple genetic steps in the progression of head and neck cancer and identifies several putative tumor suppressor loci on affected chromosomes.

Progression of basal cell carcinoma through loss of chromosome 9q and inactivation of a single p53 allele.

Basal cell carcinoma (BCC) of the skin represents a unique group of tumors strongly associated with exposure to UV light. Unlike squamous carcinoma of the skin, BCC is generally indolent, noninvasive, and rarely metastatic. To study the involvement of tumor suppressor genes in these neoplasms, we analyzed 36 BCCs for p53 mutations and a subset of these tumors for loss of chromosomes 17p and 9q. Sixty-nine % of sporadic BCCs had lost a 9q allele, with the common area of loss surrounding the putative gene for nevoid BCC or Gorlin's syndrome. Forty-four % (16 of 36) of BCCs had a mutated p53 allele, usually opposite pyrimidine tracts, which is consistent with UV-induced mutations. Surprisingly, only one tumor had lost a 17p allele, and in all BCCs only one p53 allele was inactivated. This is in direct contrast to other epithelial tumors, which usually progress by the inactivation of both p53 alleles.

Microsatellite instability in bladder cancer.

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT)n repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG)n repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (> 2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis.

Evidence for two bladder cancer suppressor loci on human chromosome 9.

Most carcinomas of the bladder show loss of heterozygosity for markers on human chromosome 9, which suggests that one or more tumor suppressor genes are located on this chromosome. Several observations suggest that such alterations are an important early step in tumorigenesis. We analyzed the pattern of allelic loss in 46 primary carcinomas of the bladder using 19 polymorphic markers from chromosome 9. While most tumors with allelic loss showed loss of heterozygosity for all informative markers that were tested, six tumors demonstrated only partial loss of chromosome 9. Two tumors with partial loss contained deletions that predominantly involved the q arm, as shown by previous studies. The other four tumors contained deletions that predominantly or exclusively involved the p arm, with a common region of loss between D9S161 (9p21) and the telomere. The results show that there is no single common region of loss on chromosome 9 and identify two distinct regions of loss that may contain bladder tumor suppressor loci.

The incidence of p53 mutations increases with progression of head and neck cancer.

To establish a genetic model of the progression of head and neck squamous carcinoma we have defined the incidence and timing of p53 mutations in this type of cancer. We sequenced the conserved regions of the p53 gene in 102 head and neck squamous carcinoma lesions. These included 65 primary invasive carcinomas and 37 noninvasive archival specimens consisting of 13 severe dysplasias and 24 carcinoma in situ lesions. The incidence of p53 mutations in noninvasive lesions was 19% (7/37) and increased to 43% (28/65) in invasive carcinomas. These data suggest that p53 mutations can precede invasion in primary head and neck cancer. Furthermore, the spectrum of codon hotspots is similar to that seen in squamous carcinoma of the lung and 64% of mutations are at G nucleotides, implicating carcinogens from tobacco smoke in the etiology of head and neck squamous carcinoma.

Inhibition of DNA replication factor RPA by p53.

The tumour suppressor p53 specifically interferes with the onset of S phase. The mechanism of the growth suppression action of the protein is unclear, though recent evidence points to transcriptional activation and repression functions of the protein. A competing hypothesis suggests that p53 interacts with the DNA replication apparatus and directly interferes with DNA replication. The major evidence for this hypothesis is that p53 interacts with the simian virus 40 (SV40)-encoded protein T antigen and interferes with the ability of T antigen to unwind the SV40 origin of DNA replication, and recruit DNA polymerase alpha to the replication initiation complex. Here we report that p53 physically interacts with and inhibits the function of a cellular DNA replication factor, the single-stranded DNA-binding protein complex RPA.

Analysis of a protein-binding domain of p53.

The tumor suppressor protein p53 was first isolated as a simian virus 40 large T antigen-associated protein and subsequently was found to function in cell proliferation control. Tumor-derived mutations in p53 occur predominantly in four evolutionarily conserved regions spanning approximately 50% of the polypeptide. Previously, three of these regions were identified as essential for T-antigen binding. We have examined the interaction between p53 and T antigen by using Escherichia coli-expressed human p53. By a combination of deletion analysis and antibody inhibition studies, a region of p53 that is both necessary and sufficient for binding to T antigen has been localized. This function is contained within residues 94 to 293, which include the four conserved regions affected by mutation in tumors. Residues 94 to 293 of p53 were expressed in both wild-type and mutant forms. T-antigen binding was unaffected by tumor-derived mutations which have been associated with the wild-type conformation of p53 but was greatly reduced by mutations which were previously shown to alter p53 conformation. Our results show that, like T-antigen binding to the Rb tumor suppressor protein, T antigen appears to interact with the domain of p53 that is commonly mutated in human tumors.

Structural alterations of the epidermal growth factor receptor gene in human gliomas.

The epidermal growth factor receptor (EGFR) gene is amplified in 40% of malignant gliomas, and the amplified genes are frequently rearranged. We have characterized the genetic alterations associated with these rearrangements in five malignant gliomas. In one tumor the rearrangement resulted in the deletion of most of the extracytoplasmic domain of the receptor, resulting in a hybrid mRNA between new sequences and the truncated EGFR sequence. The predicted amino acid sequence of the protein from this tumor was remarkably similar to that described for several viral erbB oncogenes. Four other tumors were noted to have internal deletions of the EGFR gene. These rearrangements brought about in-frame deletions affecting either of two cysteine-rich domains in the extracytoplasmic portion of the molecule. The clonal nature of these alterations, and the fact that identical alterations were seen in more than one tumor, suggests a role for these mutant receptor proteins in tumorigenesis. Further, these studies document the existence of tumor-specific cell surface molecules resulting from somatic mutation.