Involvement of Lyn and the atypical kinase SgK269/PEAK1 in a basal breast cancer signaling pathway.

Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal-regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference-mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention.

Imprinted genes that regulate early mammalian growth are coexpressed in somatic stem cells.

Lifelong, many somatic tissues are replenished by specialized adult stem cells. These stem cells are generally rare, infrequently dividing, occupy a unique niche, and can rapidly respond to injury to maintain a steady tissue size. Despite these commonalities, few shared regulatory mechanisms have been identified. Here, we scrutinized data comparing genes expressed in murine long-term hematopoietic stem cells with their differentiated counterparts and observed that a disproportionate number were members of the developmentally-important, monoallelically expressed imprinted genes. Studying a subset, which are members of a purported imprinted gene network (IGN), we found their expression in HSCs rapidly altered upon hematopoietic perturbations. These imprinted genes were also predominantly expressed in stem/progenitor cells of the adult epidermis and skeletal muscle in mice, relative to their differentiated counterparts. The parallel down-regulation of these genes postnatally in response to proliferation and differentiation suggests that the IGN could play a mechanistic role in both cell growth and tissue homeostasis.

Loss of E2F7 expression is an early event in squamous differentiation and causes derepression of the key differentiation activator Sp1.

Squamous differentiation is controlled by key transcription factors such as Sp1 and E2F. We have previously shown that E2F1 can suppress transcription of the differentiation-specific gene, transglutaminase type 1 (TG1), by an indirect mechanism mediated by Sp1. Transient transfection of E2F1-E2F6 indicated that E2F-mediated reduction of Sp1 transcription was not responsible for E2F-mediated suppression of squamous differentiation. However, we found that E2F4 and E2F7, but not E2Fs 1, 2, 3, 5, or 6, could suppress the activation of the Sp1 promoter in differentiated keratinocytes (KCs). E2F4-mediated suppression could not be antagonized by E2Fs 1, 2, 3, 5, or 6 and was localized to a region of the human Sp1 promoter spanning -139 to + 35 bp. Chromatin immunoprecipitation analysis, as well as transient overexpression and short hairpin RNA knockdown experiments indicate that E2F7 binds to a unique binding site located between -139 and -119 bp of the Sp1 promoter, and knockdown of E2F7 in proliferating KCs leads to a derepression of Sp1 expression and the induction of TG1. In contrast, E2F4 knockdown in proliferating KCs did not alter Sp1 expression. These data indicate that loss of E2F7 during the initiation of differentiation leads to the derepression of Sp1 and subsequent transcription of differentiation-specific genes such as TG1.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation.

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.

Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases.

We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.

Cortactin modulates RhoA activation and expression of Cip/Kip cyclin-dependent kinase inhibitors to promote cell cycle progression in 11q13-amplified head and neck squamous cell carcinoma cells.

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) and inhibition of S-phase entry. These effects were associated with increased binding of p21(WAF1/Cip1) and p27(Kip1) to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21(WAF1/Cip1) and p27(Kip1) at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27(Kip1) and p57(Kip2) for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

Tyrosine phosphorylation profiling reveals the signaling network characteristics of Basal breast cancer cells.

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry-based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup.

Tumor-initiating activity and tumor morphology of HNSCC is modulated by interactions between clonal variants within the tumor.

Tumor initiation (TI) in xenotransplantation models of head and neck squamous cell carcinoma (HNSCC) is an inefficient process. Poor TI could be due to (1) posttransplant cell loss, (2) a rare sub-population of cancer stem cells or (3) a requirement for specific cellular interactions, which rely on cell number. By tracking GFP-expressing HNSCC cells, we conclude that the posttransplant loss of cancer cells is minimal in the xenotransplant model. Furthermore, an examination of putative cancer stem cell markers (such as CD133, CD44, SP and label retention) in HNSCC cell lines revealed no correlation between marker expression and tumorigenicity. In addition, single-cell clones randomly isolated from HNSCC cell lines and then transplanted into mice were all capable of initiating tumors with efficiencies varying almost 34-fold. As the observed variation in the clones was both more and less tumorigenic than the parental cells, a combination of two clones, at suboptimal cell numbers for TI, was implanted into mice and was found to modulate the tumor-initiating activity, thus indicating that TI is dependent on a 'critical' number of cells and, for the first time, that interactions between clonal variants within tumors can modulate the overall tumor-initiating activity. Put in context with previous literature on tumorigenic activity, we believe that interactions between clonal variants within a tumor as well as (1) stromal interactions, (2) angiogenic activity, (3) immunocompetence and (4) cancer stem cells may all contribute to tumorigenic potential and the propensity for tumor growth and recurrence.

E2F7 can regulate proliferation, differentiation, and apoptotic responses in human keratinocytes: implications for cutaneous squamous cell carcinoma formation.

The E2F family of transcription factors plays a crucial role in the regulation of genes involved in cell proliferation, differentiation, and apoptosis. In keratinocytes, the inhibition of E2F is a key step in the control and initiation of squamous differentiation. Because the product of the recently identified E2F7a/E2F7b gene has been shown to repress E2F-regulated promoters, and to be abundant in skin, we examined its role in the epidermis. Our results indicate that E2F7b mRNA expression is selectively associated with proliferation-competent keratinocytes. Moreover, E2F7 was able to antagonize E2F1-induced proliferation and apoptosis. In contrast, although E2F7 was able to inhibit proliferation and initiate differentiation, it was unable to antagonize the differentiation suppression induced by E2F1. These data indicate that E2F7-mediated suppression of proliferation and apoptosis acts through E2F1-dependent pathways, whereas E2F7-induced differentiation acts through an E2F1-independent pathway. These data also suggest that proliferation, differentiation, and survival of primary human keratinocytes can be controlled by the relative ratio of E2F1 to E2F7. Because deregulated proliferation, differentiation, and apoptosis are hallmarks of cancer, we examined the expression levels of E2F1 and E2F7 in cutaneous squamous cell carcinomas (CSCC). We found that both genes were overexpressed in CSCCs compared with normal epidermis. Furthermore, inhibition of E2F7 in a SCC cell line sensitized the cells to UV-induced apoptosis and doxorubicin-induced apoptosis. Combined, these data suggest that the selected disruption of E2F1 and E2F7 in keratinocytes is likely to contribute to CSCC formation and may prove to be a viable therapeutic target.

Valproic acid as a therapeutic agent for head and neck squamous cell carcinomas.

PURPOSES: Here we investigate if valproic acid (VA) can enhance the efficacy of commonly used therapies for head and neck squamous cell carcinomas (HNSCC) and the molecular mechanisms that may be related to its anticancer effects. METHODS: Proliferation and viability of distinct cell types subjected to VA treatment alone or in combination regimens were measured through BrdU incorporation and LDH release, respectively. Molecular markers compatible with histone deacetylase inhibitory activity of VA were assessed through western blots assays in lysates obtained from cultured cells and tumour biopsies. RESULTS: Treatment of all cell types with VA resulted in a dose-dependent increase in histone H3 acetylation and p21 expression, as well as dose-dependent cytostasis. In contrast, the cytotoxic response to VA was variable and did not correlate with cytostasis, histone acetylation or p21 induction. The variability in response to VA was also observed in tumour biopsy samples collected from patients prior to and following a 1 week oral course of VA. In addition, we found that a combination of a clinically achievable concentration of VA plus cisplatin caused a threefold to sevenfold increase in cisplatin cytotoxicity in vitro. CONCLUSIONS: VA acts as a histone deacetylase inhibitor (HDI) in SCC cells and normal human keratinocytes (HKs), potentiates the cytotoxic effect of cisplatin in SCC cell lines and decreases the viability of SCC cells as opposed to HKs. Taken together, the results provide initial evidence that VA might be a valuable drug in the development of better therapeutic regimens for HNSCC.