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Breast cancer (BCa) is the most prevalent type of cancer in women. Several therapies used in the treatment of breast cancer are associated with clinically important rates of cardiovascular toxicity during or after treatment exposure, including anthracyclines. There is a need for new BCa therapeutics and treatments that mitigate chemotherapy-induced cardiotoxicity in BCa. In this study, we examine the effects of Serine/Threonine Kinase 3 (STK3) inhibition in the context of BCa therapy and cardioprotection from doxorubicin. STK3 (also known as MST2) is a key member of the Hippo Tumor-Suppressor Pathway, which regulates cell growth and proliferation by inhibiting YAP/TAZ co-transcription factors. Canonically, STK3 should restrict BCa growth; however, we observed that STK3 is amplified in BCa and associated with worse patient outcomes, suggesting a noncanonical pro-tumorigenic role. We found BCa cell lines have varying dependence on STK3. SUM52PE cells had the highest expression and dependence on STK3 in genetic and pharmacological assays. MCF-7 and MDA-MB-231 were less sensitive to STK3 targeting in standard proliferation assays, but were STK3 dependent in colony formation and matrigel invasion assays. In contrast, STK3 inhibition mitigated the toxic effects of doxorubicin in H9C2 rat cardiomyocytes by increasing YAP expression. Importantly, STK3 inhibition in BCa cells did not interfere with the therapeutic effects of doxorubicin. Our studies highlight STK3 is a potential molecular target for BCa with dual therapeutic effects: suppression of BCa growth and progression, and chemoprotection in cardiomyocytes.

The figures depicts the optimization of PFO-D4 cholesterol probe

Supplemental Table 1. Adjusted p-Values of SH-4-54 and 27HC (10 uM) single and combination treatment by 2Way ANOVA with Tukey's Multiple Comparisons Posthoc Statistical Analysis Supplemental Table 2. Adjusted p-Values of C188-9 and 27HC (10 uM) single and combination treatment by 2Way ANOVA with Tukey's Multiple Comparisons Posthoc Statistical Analysis

Reagents and cell culture conditions. Methods for bioinformatics analysis, CYP27A1 IHC, caspase assays and 27HC measurements.

Evaluation of CYP27A1 expression in PC cells

Table S1. CYP27A1 transcript is down-regulated in primary prostate tumors. Table S2. CYP27A1 transcript is down-regulated in metastatic PC relative to primary tumors. Table S3. CYP27A1 transcript levels in hormone sensitive PC relative to hormone resistant PC

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662–73. ©2017 AACR.

Supplementary Figure 3 from Expression of CDK4 or CDK2 in Mouse Oral Cavity Is Retained in Adult Pituitary with Distinct Effects on Tumorigenesis

Supplementary Figure 1 from Expression of CDK4 or CDK2 in Mouse Oral Cavity Is Retained in Adult Pituitary with Distinct Effects on Tumorigenesis

Supplementary Figures 1-4 from Role of Cyclin D1 as a Mediator of c-Met– and β-Catenin–Induced Hepatocarcinogenesis

Activation of c-Met signaling and β-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated β-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of β-catenin. To determine the importance of CCND1 as a mediator of c-Met– and β-catenin–induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, β-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by β-catenin/c-Met. In addition, when activated β-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of β-catenin during HCC pathogenesis, although other molecules may be required to fully propagate β-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and β-catenin. [Cancer Res 2009;69(1):253–61]

Hair follicles regenerate periodically by spontaneously undergoing cycles of growth, regression, and relative quiescence. During the hair cycle, follicle stem cells residing in a specialized niche remain quiescent, and they are stimulated to proliferate throughout the growth phase of the hair follicle. Although cell cycle regulators play a prominent role during the activation of hair follicle stem cells, the identity and the role of these regulators have not been confirmed. Herein, we reported that stem cells located in the bulge region of the HF (BuSCs) express high levels of cyclin-dependent kinase 4 (CDK4) through the quiescent phase of the hair cycle. Using gain- and loss-of-function studies, we have determined that the CDK4 protein level affects the number of BuSCs. Transgenic expression of CDK4 in the bulge region of the hair follicles reduces the number of BuSCs, whereas CDK4 ablation resulted in an increasing number of BuSCs. These results suggest that deregulation of CDK4 protein levels contributes to distorting the self-renewal/proliferation balance and, in turn, altering the number of BuSCs.

We recently reported that restoring the CYP27A1–27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti–prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6–JAK–STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6–JAK–STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation. Implications: Collectively, these data show that modulation of intracellular cholesterol by 27HC can inhibit IL6–JAK–STAT signaling and may synergize with STAT3-targeted compounds.

Current advancements in prostate cancer (PC) therapies have been successful in slowing PC progression and increasing life expectancy; however, there is still no curative treatment for advanced metastatic castration resistant PC (mCRPC). Most treatment options target the androgen receptor, to which many PCs eventually develop resistance. Thus, there is a dire need to identify and validate new molecular targets for treating PC. We found NUAK family kinase 2 (NUAK2) expression is elevated in PC and mCRPC versus normal tissue, and expression correlates with an increased risk of metastasis. Given this observation and because NUAK2, as a kinase, is actionable, we evaluated the potential of NUAK2 as a molecular target for PC. NUAK2 is a stress response kinase that also plays a role in activation of the YAP cotranscriptional oncogene. Combining pharmacological and genetic methods for modulating NUAK2, we found that targeting NUAK2 in vitro leads to reduction in proliferation, three-dimensional tumor spheroid growth, and matrigel invasion of PC cells. Differential gene expression analysis of PC cells treated NUAK2 small molecule inhibitor HTH-02-006 demonstrated that NUAK2 inhibition results in downregulation of E2F, EMT, and MYC hallmark gene sets after NUAK2 inhibition. In a syngeneic allograft model and in radical prostatectomy patient derived explants, NUAK2 inhibition slowed tumor growth and proliferation rates. Mechanistically, HTH-02-006 treatment led to inactivation of YAP and the downregulation of NUAK2 and MYC protein levels. Our results suggest that NUAK2 represents a novel actionable molecular target for PC that warrants further exploration.

All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main stress-coping mechanism is the stringent response triggered by the accumulation of “alarmone” (p)ppGpp to arrest proliferation and reprogram transcriptome. While mammalian genomes encode MESH1—the homolog of the (p)ppGpp hydrolase SpoT, current knowledge about its function remains limited. We found MESH1 expression tended to be higher in tumors and associated with poor patient outcomes. Consistently, MESH1 knockdown robustly inhibited proliferation, depleted dNTPs, reduced tumor sphere formation, and retarded xenograft growth. These antitumor phenotypes associated with MESH1 knockdown were accompanied by a significantly altered transcriptome, including the repressed expression of TAZ, a HIPPO coactivator, and proliferative gene. Importantly, TAZ restoration mitigated many anti-growth phenotypes of MESH1 knockdown, including proliferation arrest, reduced sphere formation, tumor growth inhibition, dNTP depletion, and transcriptional changes. Furthermore, TAZ repression was associated with the histone hypo-acetylation at TAZ regulatory loci due to the induction of epigenetic repressors HDAC5 and AHRR. Together, MESH1 knockdown in human cells altered the genome-wide transcriptional patterns and arrested proliferation that mimicked the bacterial stringent response through the epigenetic repression of TAZ expression.

Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo Tumor suppressor pathway which regulates Yes 1 Associated protein (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulate YAP1/TAZ activation and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyper-activation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. A high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth and matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo and biochemical analysis suggest a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemo-protective effects. Our results contend that STK3 has a non-canonical role in PC progression and inhibition of STK3 may have therapeutic potential for PC that merits further investigation.SignificanceOur findings illuminate a new actionable target for PC therapy that would traditionally be overlooked due to its canonical role as a tumor suppressor in other cancer types.

BACKGROUND: Some, but not all, epidemiologic evidence supports a role for cholesterol, the precursor for steroid hormone synthesis, in prostate cancer. Using a PTEN-null transgenic mouse model of prostate cancer, we tested the effect of modifying serum cholesterol levels on prostate tumor development and growth. We hypothesized that serum cholesterol reduction would lower tumor androgens and slow prostate cancer growth.METHODS: PTENloxP/loxP-Cre+ mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (30 mg/kg/day), or no intervention, and sacrificed at 2, 3, or 4 months of age. Serum cholesterol and testosterone were measured by ELISA and intraprostatic androgens by mass spectrometry. Prostate histology was graded, and proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL, respectively.RESULTS: Ezetimibe-treated mice had lower serum cholesterol at 4 months (p = 0.031). Serum cholesterol was positively correlated with prostate weight (p = 0.033) and tumor epithelial proliferation (p = 0.069), and negatively correlated with tumor epithelial apoptosis (p = 0.004). Serum cholesterol was unrelated to body weight (p = 0.195). Tumor stromal cell proliferation was reduced in the ezetimibe group (p = 0.010). Increased serum cholesterol at 4 months was associated with elevated intraprostatic DHEA, testosterone, and androstenedione (p = 0.043, p = 0.074, p = 0.031, respectively). However, cholesterol reduction did not significantly affect adenocarcinoma development at 2, 3, or 4 months of age (0, 78, and 100% in ezetimibe-treated vs. 0, 80, and 100% in mice not receiving ezetimibe).CONCLUSIONS: Though serum cholesterol reduction did not significantly affect the rate of adenocarcinoma development in the PTEN-null transgenic mouse model of prostate cancer, it lowered intraprostatic androgens and slowed tumor growth. These findings support a role for serum cholesterol in promoting prostate cancer growth, potentially via enhanced tumor androgen signaling, and may provide new insight into cholesterol-lowering interventions for prostate cancer treatment.

Prostate cancer (PC) is the second cause of cancer related deaths in men. Most therapies target the Androgen Receptor (AR) which can prolong survival, but resistance emerges and patients eventually succumb to the disease. This highlights the need for new PC molecular targets. Our group observed in PC patient datasets, the Serine/threoninekinase3 (STK3) gene is frequently amplified and correlates with worse outcomes. This is against the established dogma that STK3 is a tumor suppressor in the Hippo Tumor suppressor pathway that canonically regulates oncogenic transcription factor YAP1. Counter to this role, we report that in PC, STK3 has a pro-tumorigenic role. Our work shows that genetic and chemical inhibition of STK3 in PC cells in vitro significantly slowed proliferation and protasphere growth. The goal of this study was to identify and validate novel STK3 kinase inhibitors with increased potency and specificity. Approach. We screened large assay panels of kinase inhibitors (ChEMBL) for STK3 active molecules, which identified two independent scaffolds. For assessment of STK3 inhibition, we utilized a battery of in cell and biochemical assays including western blot, STK3-NanoBRET assays, radiometric enzymatic assays and a broad KINOMEscan scanMax panels to assess leads and analogs. For phenotypic studies, we utilized Hi-myc ventral prostate (HMVP2) cells to test STK3 inhibition in 3D spheroid growth and spheroid invasion of matrigel by live cell imaging. Pharmacokinetics (PK) and pharmacodynamics (PD) were performed for oral (PO) and intraperitoneal (IP) routes of our top STK3 inhibitor. A subcutaneous HMVP2 allograft model was used to test in vivo efficacy of STK3 inhibition by daily IP injections at 10 mg/kg and 30 mg/kg doses vs. vehicle. Tumor volumes were tracked by caliper measurements and at endpoint, we measured tumor, liver, lung, heart and spleen weights. Results. The two lead scaffolds and a number of analogs showed significant inhibition of STK3 by western blot analysis, in cell STK3-NanoBRET assay and recombinant STK3 radiometric assays. Phenotypic studies in HMVP2 spheres treated with analog STK3 inhibitors showed dose dependent decrease in proliferation and matrigel invasion. From our assays, compound SOB-5-47 emerged as the most potent. PK and PD studies showed that IP injections of SOB-5-47 led to short time in plasma, but pro-longed half-life in the organs such as liver, heart and notably prostate with a terminal half-life clearance of 8.3hrs. Daily injections of SOB-5-47 at 10 and 30 mgs/kg significantly delayed HMVP2 tumor kinetics compared to vehicle control. Tumor weights in SOB-5-47 groups were significantly lower vs control. Importantly, no differences in organ weights or total body weights were observed between groups. Conclusions. Here we report a noncanical role of STK3 in PC and the development of novel STK3 kinase inhibitors that can be used as tools to further examine the role of STK3. Additionally, we show that STK3 can be targeted in vivo to slow PC growth without significant unwanted deleterious effects. Citation Format: Amelia U. Schirmer, Lucy M. Driver, Megan T. Zhao, Carrow I. Wells, Xuan Yang, David H. Drewry, Ivan Spasojevic, Everardo Macias. Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P149.

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662–73. ©2017 AACR.

OBJECTIVES: Carbohydrates are the main source of energy in older adults in the US. Moreover, they increase insulin and insulin-like growth factor-1 (IGF1), which are implicated in tumor growth by increasing cell survival. Previously, we found low carbohydrate (LC) diets slow prostate cancer (PC) growth and increase survival vs. a Western diet (WD) in mice. However, long-term adherence to a LC diet can be difficult for cancer patients. Thus, we aimed to determine whether modifying carbohydrate quality without changing quantity could result in the same outcome as when quantity is reduced. Carbohydrate quality was based on a glycemic index (GI), which indicates how carbohydrates affect blood glucose levels. Low GI carbohydrates are absorbed more slowly and result in a lower and slower increase in glucose levels and insulin demand than high GI carbohydrates. We hypothesized that high carbohydrate intake but with a low GI would slow PC growth, by reducing insulin levels. METHODS: A xenograft mice study compared the effect on PC growth of 3 diets: HiGI WD (48% carbohydrate: sucrose), HiGI LC (20% carbohydrate: sucrose), and LoGI WD (48% carbohydrate: amylose, amylopectin, maltodextrin). Male SCID mice were fed an ad lib HiGI WD. At day 14, they were injected with 5 × 10(5) LAPC-4 cells. When tumors reached ∼200 mm(3), mice were single-housed and randomized to their diets (n = 33/group). Mice were pair-fed to ensure all had the same weight throughout the study. Tumor volume and body weight were measured 2X per week. Body composition was determined by Echo-MRI. The outcomes of the study were tumor volume, survival, glucose, insulin, IGF-1 and IGFBP-3 levels, and tumor tissue analysis. RESULTS: LoGI WD mice had lower tumor volumes, insulin, IGF1, and IGF1: IGFBP3 ratio, and higher IGF-BP3 levels, than the other two groups. Blood glucose was similar across arms. Even though body weight was similar across arms, LoGI WD mice had lower body fat. In a meal tolerance test, glucose was higher in HiGI WD mice with comparable results between the other two diets. CONCLUSIONS: Feeding mice a low GI diet delayed PC growth and decreased serum insulin, IGF1 and IGF1: IGFBP3 ratios vs. a high GI diet. These data suggest carbohydrate quality is important for PC growth. Whether a low-carbohydrate and low GI diet would have additive benefits remains to be tested. FUNDING SOURCES: American Institute for Cancer Research.

BACKGROUND: Previously, we showed that carbohydrate restriction with calorie restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction without calorie restriction on tumor development within the context of diet-induced obesity in the Hi-Myc transgenic mouse model of prostate cancer.METHODS: Mice were randomized at 5 weeks of age to ad libitum western diet (WD; 40% fat, 42% carbohydrate; n=39) or ad libitum no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n=44). At age 3 or 6 months, mice were killed, prostates weighed and prostate histology, proliferation, apoptosis and macrophage infiltration evaluated by hematoxylin and eosin, Ki67, TUNEL and F4/80 staining, respectively. Body composition was assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western.RESULTS: Caloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age, relative to the WD group (45 g vs 38g; P=0.008). Despite elevated body weights, serum monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1α levels were lower in NCKD versus WD mice (P=0.046 and P=0.118, respectively), and macrophage infiltration was reduced in prostates of NCKD versus WD mice (P=0.028). Relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD versus WD mice. However, while mice randomized to NCKD had smaller prostates after adjustment for body weight at 3 and 6 months (P=0.004 and P=0.002, respectively), NCKD mice had higher rates of adenocarcinoma at 6 months compared to WD mice (100 vs 80%, P=0.04).CONCLUSIONS: Despite higher caloric intake and elevated body weights, carbohydrate restriction lowered serum MCP-1 levels, reduced prostate macrophage infiltration, reduced prostate weight, but failed to slow adenocarcinoma development. Together, these data suggest that although carbohydrate restriction within the context of obesity may reduce obesity-associated systemic inflammation and perhaps slow tumor growth, it is not sufficient to counteract obesity-associated tumor development.

BACKGROUND Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts. METHODS Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 105 LAPC-4 cells. Two weeks post-injection, mice were randomized to control, 11 mg/kg/day simvastatin, 30 mg/kg ezetimibe, or the combination and sacrificed 42 days post-randomization. We used a generalized linear model with the predictor variables of treatment, time, and treatment by time (i.e., interaction term) with tumor volume as the outcome variable. Total serum and tumor cholesterol were measured. Tumoral RNA was extracted and cDNA synthesized from 1 ug of total RNA for quantitative real-time PCR. RESULTS Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors. CONCLUSIONS Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate © 2016 Wiley Periodicals, Inc.

Obesity is a US and global epidemic that exacerbates various cancers including prostate cancer (PC). A recent meta-analysis of men with PC found a 20% increased risk of PC-specific mortality for each 5 kg/m2 increase in BMI. To identify ways to exploit this, we performed an in vivo screen to identify kinases that were causally involved in tumor/growth in obese mice. This resulted in the identification of the AMPK-related kinase SNARK a.k.a NUAK2, a YAP1 target gene and positive feedback regulator by inhibition LATS1 kinase. To identify obesity essential kinases LAPC-4 cells were inoculated with an shRNA library of ~5,000 lentivirus targeting 513 kinases. 5 × 106 cells were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. NAUK2 shRNAs (3 of 10) were significantly depleted in LAPC4 xenograft tumors from mice on a Western diet (WD), but not tumors from mice on an isocaloric 25% calorie-restricted (CR) diet. Analysis of NUAK2 gene expression in human PCs shows NUAK2 expression is higher in PC tissue vs. normal benign. In addition, Cox regression analysis showed that NUAK2 is associated with a 3-fold increased chance of developing metastasis disease in high NUAK2 group. Proteomic data from metastatic PC rapid autopsy biopsies shows NUAK2 phospho-target LATS1 Ser464 is higher in metastatic PCs vs. benign localized prostatic tissues. Western blot analysis of PC cell lines shows NUAK2 is elevated in comparison to primary prostate epithelial cells. To test if NUAK2 is an actionable target in PC cells, we utilized two narrow-spectrum kinase inhibitors from the literature, WZ4003 and HTH-02-006. Pharmacologic inhibition of NUAK2 slows 2D PC cell growth and 3D PC spheroid growth of various PC cell lines with an IC50 range between 1-10 uM, with HTH-02-006 being the more potent. In addition, NUAK2 kinase inhibition with WZ4003 and HTH-02-006 significantly slowed migration and Matrigel invasion of PC3 and DU145 cells. To test if NUAK2 is associated with obesity-enhanced PC progression, we tested NUAK2 inhibition in combination with palmitic acid supplementation. Our preliminary studies show that pharmacologic inhibition of NUAK2 sensitized PC cells to fatty acid-induced cell death. In conclusion, our in vivo shRNA screen identified NUAK2 as an essential kinase for tumors grown in obese mice. NUAK2 expression correlates with worse outcomes in PC and NUAK2 kinase activity is elevated in metastatic vs. localized PCs. Our preliminary data show that pharmacologic inhibition of NUAK2 slows PC cell growth and migration/invasion. Lastly, our preliminary studies show that NUAK2 may lower the threshold for fatty acid-induced toxicity. Together, these preliminary data suggest NUAK2 is a relevant actionable target in PC and may synergize with obese host physiology to slow PC tumor growth. Citation Format: Weiwei Fu, Megan Zhao, Amelia Schirmer, Erick J. Maravilla, Junhee Yoon, Sungyong You, Stephen J. Freedland, Everardo Macias. NUAK2 inhibition for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B07.

The nuclear transcription factor signal transducer and activator of transcription 3 (Stat3) has recently been reported to have a localized mitochondrial regulatory function. Current data suggest that mitochondrial Stat3 (mitoStat3) is necessary for maximal mitochondrial activity and for Ras-mediated transformation independent of Stat3 nuclear activity. We have previously shown that Stat3 has a pivotal role in epithelial carcinogenesis. Therefore, the aim of the current study was to determine the role of mitoStat3 in epidermal keratinocytes. Herein, we show that normal and neoplastic keratinocytes contain a pool of mitoStat3. EGF and 12-O-tetradecanoylphorbol-13-acetate induce Stat3 mitochondrial translocation mediated through the phosphorylation of Stat3 at Ser727. In addition, we report that mitoStat3 binds mtDNA and associates with the mitochondrial transcription factor A. Furthermore, Stat3 ablation resulted in an increase of mitochondrial-encoded gene transcripts. An increase in key nuclear-encoded metabolic genes, PGC-1α and NRF-1, was also observed in Stat3-null keratinocytes; however, no changes in nuclear-encoded electron transport chain gene transcripts or mtDNA copy number were observed. Collectively, our findings suggest a heretofore-unreported function for mitoStat3 as a potential mitochondrial transcription factor in keratinocytes. This mitoStat3–mtDNA interaction may represent an alternate signaling pathway that could alter mitochondrial function and biogenesis and have a role in tumorigenesis.

Obesity is associated with greater risk of high-grade prostate cancer (PC), recurrence after therapy, metastases, and PC specific mortality. The fact that obesity is a PC risk factor creates an opportunity to identify and target the mechanisms underlying obesity-enhanced lethal PC. We exploited this link by performing an shRNA in vivo screen in obese mice targeting the entire kinome, which found Right Open Reading Frame Kinase 2 (RIOK2) as essential for PCs in obese mice. We found RIOK2 gene signature is correlated with a 56% increased risk of metastatic PC. Moreover, when we queried only men with a BMI ≥ 30 kg/m2 we found that high levels of RIOK2 gene signature increased the risk of metastasis by an additional 50%. RIOK2, a serine/threonine kinase, is critical for de novo ribosome maturation and assembly, which is essential to maintain the high proliferation rates in cancer cells. Indeed, we found that RIOK2 loss reduced de novo protein synthesis in PC cells and decreased cell viability. Conversely, RIOK2 overexpression enhanced PC cell growth. As our goal is to find actionable PC targets to reduce obesity effects on PC, we conducted a protein structure based compound screen in silico and identified a lead kinase inhibitor of RIOK2 (kiRIOK2). Our lead kiRIOK2 inhibits cell proliferation, protein synthesis, anchorage-independent growth and invasion of multiple PC lines in the µM range. SAR optimized kiRIOK2 have increased efficacy and bioavailability. Our studies are providing probes and mechanistic insights to further investigate the role of RIOK2 in PC biology. In the long-term, this may help mitigate the added risk of obesity on PC metastasis and PC-specific mortality. Citation Format: Everardo Macias, Jiyoon Cho, Drew Rosowicz, Zahra Heidari, Sungyong You, Erick J. Maravilla, Jen-Tsan Chi, Stephen J. Freedland. Mechanisms and inhibition RIOK2 for obesity-driven prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3590.

Serine/threonine kinase 3 (STK3) and its paralog STK4, encode MST2 and MST1 kinases respectively, which are essential members of the highly conserved Hippo Tumor suppressor pathway which regulate Yes 1 Associated protein (YAP1). In normal cells YAP1 activation is beneficial for organ and tissue regeneration, with overgrowth being controlled by MST1/2. In cancer cells, MST1/2 play a role in cancer cell proliferation, differentiation and apoptosis by inhibiting YAP1, yet frequent deregulation of MST1/2 leads to hyper-activation of YAP1 in various cancers. Th current dogma is that STK3/4 are tumor suppressor genes. Yet, STK3, but not STK4 in prostate cancer (PC) is frequently amplified. The goal of this study was to determine if STK3 and/or STK4 gene products MST1/2 play a role in PC growth and/or metastasis. Approach. To assess if MST1 and/or MST2 play a role in PC proliferation, pharmacological and genetic studies were carried out across varying PC cell lines in vitro. XMU-MP-1 a narrow spectrum kinase inhibitor of MST1/2 was used across five distinct PC cell lines. We utilized 2D proliferation and 3D PC cell spheroids to assess effects through live cell image using Incuctye S3. Invasion assays were performed using a 96 well scratch wound migration with or without matrigel with PC3 and DU145 cells. We queried cbioPortal for PC data sets to look at incidence of STK3 and STK4 copy number alterations. Knockdowns of STK3 and STK4 genes using shRNAs to observe phenotypic differences and growth inhibition. Knockdown of STK3/4 pathways were verified through western blot. Results. Proliferation experiments 5 cell lines found that inhibition of STK3/4 with XMU-MP-1 significantly slows 2D and 3D spheroid PC cell growth. IC50 scores ranged from ~500nM to 5uM. Additionally, STK3/4 inhibition in the extremely invasive PC3 and DU145 cell lines significantly inhibited cell migration and matrigel invasion. PC data sets showed that the STK3 gene is frequently amplified in PC tumors which is contrary to an expected tumor suppressive role. In contrast, STK4 was not found to be amplified suggesting variation in STK3 versus STK4 roles. Depletion of STK3 and STK4 individually by shRNA knockdown also inhibited cell growth PC cells. Phenotypic markers of apoptosis and cell death could be seen 72 hours post viral transduction with shRNA knockdown in 22Rv1 and PC3 PC cells. The shRNA knockdowns showed deceased phosphorylated MOB kinase activator 1(MOB1) a downstream phospho-target of MST1/2. However, mixed results were obtained for subsequent YAP phosphorylation. Conclusions. Our results demonstrate that STK3/4 are essential for PC cell proliferation and migration/invasion. Similar effects were observed by both inhibition of MST1/2 either directly through drug treatment or STK3/4 gene depletion by shRNA knockdown. Whether these paralogs have unique or overlapping effects and if they can be targeted for PC therapy remains to be elucidated. Citation Format: Amelia Schirmer, Alexis B. Stokes, Erick J. Maravilla, Weiwei Fu, Everardo Macias. Inhibition of Hippo tumor suppressors MST1/2 slows prostate cancer cell proliferation and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 862.

The current study was designed to explore the role of signal transducer and activator of transcription 1 (Stat1) during tumor promotion using the mouse skin multistage carcinogenesis model. Topical treatment with both 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3-methyl-1,8-dihydroxy-9-anthrone (chrysarobin or CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Y701) and serine (S727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. CHRY treatment also led to upregulation of interferon regulatory factor 1 (IRF-1) mRNA and protein, which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNγ) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNγ signaling. Stat1 deficient (Stat1(-/-) ) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1(-/-) mice and wild-type littermates with TPA as the promoter. Maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNγ signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1, IRF-1 and uStat1.

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation and migration as well as in cancer development. In the present study, we examined the impact of constitutive activation of Stat3 on behavior of keratinocytes, including keratinocyte stem cells (KSC) in vivo. BK5.Stat3C transgenic (Tg) mice, which express a constitutively active form of Stat3 (Stat3C) in the basal layer of the epidermis and in the bulge region KSCs exhibited a significantly reduced number of CD34+/α6 integrin+ cells compared to non-transgenic (NTg) littermates. There was a concomitant increase in the Lgr-6, Lrig-1, and Sca-1 populations in the Tg mice in contrast to the CD34 and Keratin-15 positive population. In addition, increased expression of c-myc, β-catenin, and epithelial–mesenchymal transition (EMT)-related genes as well as decreased expression of α6-integrin was observed in the hair follicles of Tg mice. Notably, Sca-1 was found to be a direct transcriptional target of Stat3 in keratinocytes. The current data suggest that elevated Stat3 activity leads to depletion of hair follicle KSCs along with a concomitant increase of stem/progenitor cells above the bulge region. Overall, the current data indicate that Stat3 plays an important role in keratinocyte stem/progenitor cell homeostasis.

Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts.Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 10Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors.Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate 77:446-457, 2017. © 2016 Wiley Periodicals, Inc.

synthesis, in prostate cancer progression. We determined the impact of modifying serum cholesterol levels on tumor progression in PTENdeficient transgenic mouse model of prostate cancer. METHODS: PTEN-Cre mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (Zetia; 30 mg/kg diet), or no intervention, and sacrificed at 2, 3, or 4 months of age. At sacrifice, prostates were removed, weighed, and fixed in formalin. Serum cholesterol and testosterone levels were measured by ELISA and prostate androgens measured by mass spectrometry. Proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL assay, respectively. RESULTS: Mice randomized to Zetia had lower serum cholesterol levels (p1⁄40.031). Cholesterol levels were positively correlated with prostate weight (p1⁄40.033) and tumor epithelial proliferation (p1⁄40.069), and negatively correlated with tumor epithelial apoptosis (p1⁄40.004). Tumor stromal cell proliferation was reduced in the Zetia group (p1⁄40.010). Increased cholesterol levels were associated with elevated intraprostatic DHEA, testosterone and androstendione (p1⁄40.043, p1⁄40.074, p1⁄40.031, respectively) but was unrelated to serum androgens (p1⁄40.08). Mice consuming Zetia diet had lower rates of intracystic adenocarcinoma at 2 and 3 months of age (0% vs. 14% and 30% vs. 44%, respectively). CONCLUSIONS: Using a PTEN-deficient transgenic mouse model of prostate cancer, we found that serum cholesterol reduction lowered intraprostatic androgen concentrations and slowed prostate cancer progression, supporting a potentially important role for cholesterol in prostate cancer. This study may provide new insight into the use of cholesterol-lowering interventions for prostate cancer prevention and treatment.

The proto-oncogene c-Myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis. Myc accelerates the rate of cell proliferation, at least in part, through its ability to down-regulate the expression of the cell cycle inhibitor p27 Kip1 . Moreover, p27 Kip1 protein levels are regulated by ubiquitin-mediated turnover, leading to destruction by the E3 ubiquitin ligase SCF Skp2 . Therefore, we hypothesize that a lack of Skp2 expression should lead to increased p27 Kip1 levels and further inhibition of Myc -mediated proliferation and tumorigenesis. Myc expression in epithelial tissues of transgenic mice (K5- Myc ) led to increased keratinocyte proliferation and the development of spontaneous tumors within the oral cavity. We generated K5- Myc –transgenic mice in an Skp2-null background. Consistent with our hypothesis, we found that Myc -mediated keratinocyte hyperproliferation was abolished by the loss of Skp2. However, Skp2 ablation did not affect Myc -driven tumorigenesis because the incidence, latency, and degree of differentiation of oral tumors were identical between K5- Myc /Skp2 +/+ and K5- Myc /Skp2 −/− mice. Altogether, these findings suggest that Skp2 and p27 Kip1 are critical for Myc -driven keratinocyte proliferation; however, Myc -mediated tumorigenesis in the oral epithelium is independent of the Skp2-p27 Kip1 axis.

In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1, suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.

Activation of c-Met signaling and β-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated β-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of β-catenin. To determine the importance of CCND1 as a mediator of c-Met– and β-catenin–induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, β-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by β-catenin/c-Met. In addition, when activated β-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of β-catenin during HCC pathogenesis, although other molecules may be required to fully propagate β-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and β-catenin. [Cancer Res 2009;69(1):253–61]

It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21Cip1 and p27Kip1. Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4D158N mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4D158N, but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21 Cip1 in K5Cdk2, but not in K5Cdk4D158N, epidermis, suggesting that CDK2 overexpression elicits a p21Cip1 response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis. Copyright © American Society for Investigative Pathology. Fil: Macias, Everardo. North Carolina State University; Estados Unidos Fil: Miliani De Marval, Paula L.. Duke University Medical Center; Estados Unidos Fil: de Siervi, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institutes of Health; Estados Unidos Fil: Conti, Claudio J.. the M.D. Anderson Cancer Center; Estados Unidos Fil: Senderowicz, Adrian M.. National Institutes of Health; Estados Unidos. United States Food and Drug Administration; Estados Unidos Fil: Rodriguez Puebla, Marcelo L.. North Carolina State University; Estados Unidos

We have previously shown that forced expression of CDK4 in mouse skin (K5CDK4 mice) results in increased susceptibility to squamous cell carcinoma (SCC) development in a chemical carcinogenesis protocol. This protocol induces skin papilloma development, causing a selection of cells bearing activating Ha-ras mutations. We have also shown that myc-induced epidermal proliferation and oral tumorigenesis (K5Myc mice) depends on CDK4 expression. Biochemical analysis of K5CDK4 and K5Myc epidermis as well as skin tumors showed that keratinocyte proliferation is mediated by CDK4 sequestration of p27Kip1 and p21Cip1, and activation of CDK2. Here, we studied the role of CDK2 in epithelial tumorigenesis. In normal skin, loss of CDK2 rescues CDK4-induced, but not myc-induced epidermal hyperproliferation. Ablation of CDK2 in K5CDK4 mice results in decreased incidences and multiplicity of skin tumors as well as malignant progression to SCC. Histopathologic analysis showed that K5CDK4 tumors are drastically more aggressive than K5CDK4/CDK2−/− tumors. On the other hand, we show that CDK2 is dispensable for myc-induced tumorigenesis. In contrast to our previous report of K5Myc/CDK4−/−, K5Myc/CDK2−/− mice developed oral tumors with the same frequency as K5Myc mice. Overall, we have established that ras-induced tumors are more susceptible to CDK2 ablation than myc-induced tumors, suggesting that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the oncogenic pathway involved. [Cancer Res 2007;67(20):9713–20]

Loss of the cyclin-dependent kinase inhibitor p27 Kip1 leads to an overall increase in animal growth, pituitary tumors, and hyperplasia of hematopoietic organs, yet it is unknown whether all cells function autonomously in response to p27 Kip1 activity or whether certain cells take cues from their neighbors. In addition, there is currently no genetic evidence that tumor suppression by p27 Kip1 is cell-autonomous because biallelic gene inactivation is absent from tumors arising in p27 Kip1 hemizygous mice. We have addressed these questions with tissue-specific targeted mouse mutants and radiation chimeras. Our results indicate that the suppression of pars intermedia pituitary tumors by p27 Kip1 is cell-autonomous and does not contribute to overgrowth or infertility phenotypes. In contrast, suppression of spleen growth and hematopoietic progenitor expansion is a consequence of p27 Kip1 function external to the hematopoietic compartment. Likewise, p27 Kip1 suppresses thymocyte hyperplasia through a cell-nonautonomous mechanism. The interaction of p27 Kip1 loss with epithelial cell-specific cyclin-dependent kinase 4 overexpression identifies the thymic epithelium as a relevant site of p27 Kip1 activity for the regulation of thymus growth.

A number of studies have established the role of receptor tyrosine kinases (RTKs) and their downstream signaling targets, including PI3K/Akt/mTOR and Stat3, as important regulatory factors of epidermal proliferative capacity during both chemical (two-stage) and UV skin carcinogenesis. We have previously shown that total protein levels of Twist1 can be regulated by Stat3 in the two-stage skin carcinogenesis model and that nuclear localization of Twist1 was restricted to the proliferative compartment. Twist1, a protein commonly associated with the induction of EMT, has previously been shown to associate with cell cycle effectors, such as p53 and p21, as well as impact cell cycle progression. The cell cycle regulatory functions of Twist1 have been linked to p53 via phosphorylation of Twist1 at Ser42 by Akt. To investigate the role of Twist1 in cellular proliferation, primary keratinocytes were infected with lentiviral particles to create Twist1 knockdown or overexpressing cells, and were used, together with wild-type primary keratinocytes, to analyze the role of Twist1 during cell cycle progression. Western blot analysis of lysates from primary keratinocytes indicated that the expression of cell cycle regulatory proteins Cyclin E1, NF-κB, and c-Myc, coincided with the decrease or increase of Twist1. Protein levels of p21 exhibited an inverse correlation to Twist1 expression. Additionally, ChIP analysis identified Cyclin D1, Cyclin E1, c-Myc, and NF-κB as novel transcriptional targets of Twist1. To further investigate the role of Twist1, we have established transgenic mice with deletion of Twist1 in the basal compartment of the epidermis using BK5.Cre (i.e. BK5.Cre x Twistflox/flox mice). BK5.Cre x Twistflox/flox mice exhibited a significant reduction in epidermal thickness and labeling index (LI) compared to wild-type mice following treatment with TPA. In comparison to wild-type mice, acetone treated Bk5.Cre x Twistflox/flox mice also displayed a significant reduction of epidermal LI. Western blot analysis of epidermal lysates collected from BK5.Cre x Twistflox/flox mice treated with TPA demonstrated a reduction of Cyclin E1 and reduced phosphorylation of Rb at Ser795 compared to wild-type mice, further implicating the regulatory capacity of Twist1 during keratinocyte proliferation. Twist1 Ser42 phosphorylation also correlated with expression of the aforementioned cell cycle proteins. Collectively, these results, combined with other data to be presented, suggest that Twist1 plays a key role in keratinocyte proliferation during skin tumor promotion. Research supported by CA76520 and T32 ES007247. Citation Format: Jaya Srivastava, Everardo Macias, Kaoru Kiguchi, John DiGiovanni. Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5355. doi:10.1158/1538-7445.AM2014-5355

Recent studies have identified the involvement of receptor tyrosine kinases (RTKs) and their downstream signaling pathways, including MAPK, PI3K/Akt/mTOR, and Stat3, as key regulatory factors of epidermal proliferative capacity during skin carcinogenesis. Through development and study of transgenic mice with overexpression of a constitutively active form of Stat3 (Stat3C) in the basal cell layer, previous studies in the laboratory discovered that constitutive Stat3 activation accelerates tumor progression in the two-stage skin carcinogenesis model, which utilizes the tumor initiator 7,12-dimethylbenz[α]anthracene (DMBA) and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Twist1, a well-known inducer of EMT, has been shown to be regulated by Stat3 in a number of systems. Western blot analysis of epidermal protein lysates from Stat3C transgenic mice indicated a two-fold induction of Twist1 expression as compared to wild type mice. In addition, immunofluorescence staining analysis of tumors from wild type mice [papillomas and squamous cell carcinomas (SCCs)] and Stat3C mice (SCCs) showed nuclear co-localization of Twist1 and Stat3. In the present study, we investigated the activation and subsequent downstream signaling of Twist1 during TPA-induced epidermal hyperproliferation. Western blot analysis of epidermal lysates from mice treated topically with multiple applications of TPA showed an initial decrease (4-6 h) followed by a subsequent rebound of Twist1 protein at 18 hours post TPA treatment. This initial decrease and then rebound coincided with that of the cell cycle regulatory proteins cyclin D1, cyclin E1, E2F-1, and c-myc. Interestingly, basal levels of Twist1 were reduced in epidermis specific Stat3 deficient mice. Furthermore, Stat3 deficient mice treated with TPA failed to show a rebound in expression of Twist1 in the epidermis. Immunofluorescence experiments of TPA treated epidermis revealed that nuclear localization of Twist1 was restricted to the proliferative compartment. Collectively, these results, combined with other data to be presented, suggest that Stat3 regulates basal levels of Twist1. This protein is localized in the nucleus of cells found primarily in the proliferative compartment of hyperplastic epidermis and skin tumors. The current results further suggest that Twist1 may play a role in cell cycle progression in keratinocytes during tumor promotion. Research supported by CA76520. Citation Format: Jaya Srivastava, Everardo Macias, Dharanija Rao, Kaoru Kiguchi, John DiGiovanni. Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1290. doi:10.1158/1538-7445.AM2013-1290

The pRb/Cdk/cyclin/p16 pathway has been found altered in a majority of human neoplasias. In particular the Cyclin-dependent kinase 4 (CDK4) has been found mutated in familial melanoma, amplified or overexpressed in human gliomas, sporadic breast carcinomas and sarcomas. Additionally, a vast number of human cancers contain high levels of CDK4 activity due to loss of the Cdk inhibitor p16Ink4a or overexpression of its binding partner cyclin D1. We have previously demonstrated that CDK4 ablation inhibits chemically-induced mouse skin papillomas, whereas forced expression of CDK4 in mouse epidermis (K5-CDK4) accelerates malignant progression to Squamous Cell Carcinomas (SCC). However, the mechanisms by which changes in CDK4 expression levels control skin tumorigenesis have not been established. In this model topical application of DMBA and TPA results in clonal expansion of a slow cycling stem cell population localized at the bulge area of the hair follicle. Thus, we hypothesize that CDK4 deletion or overexpression affects tumorigenesis by altering the characteristic and/or the number of Keratinocytes Stem Cells (KSC). To address this, we employed the K15-EGFP transgenic mouse model, which expresses EGFP under the control of the keratin 15 promoter in the bulge area of the hair follicle, to generated K15-EGFP/K5-CDK4as well as K15-EGFP/CDK4−/− compound mice. Flow cytometry analysis of K15-EGFP/K5-CDK4 keratinocytes show 4-fold decrease in the number of KSC compared to K15-EGFP control mice. In contrast, K15-EGFP/CDK4−/− compound mice show 4-fold increase in the number of KSC compared with control siblings. Quantification of mRNA in the KSCs showed 3 to 5-fold increase in the CDK4 levels of KSC compared to non-Stem cell population, whereas mild or no changes in CDK2, Cyclin D1 and p27Kip1mRNA levels were detected. Consistent with these data, we also determined an increase number of Labeled-Retaining Cells (LRC) in the hair follicle of CDK4−/− and decreased LRC in K5-CDK4 mice. Together these results are consistent with our hypothesis that changes in CDK4 expression affects the asymmetric cell division of KSC population favoring an increase in the transit amplifying (TA) cell pool and decrease of the KSC pool in K5-CDK4 and the opposite in CDK4−/− mice. As a result, this model predicts that the number of transit amplifying or progenitor cells correlate with the susceptibility to papilloma development. Supported by NIH grant CA116328 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3889.

S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.