Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

A Phase I Study of Dinaciclib in Combination With MK-2206 in Patients With Advanced Pancreatic Cancer.

The combination of drugs targeting Ral and PI3K/AKT signaling has antitumor efficacy in preclinical models of pancreatic cancer. We combined dinaciclib (small molecule cyclin dependent kinase inhibitor with MK-2206 (Akt inhibitor) in patients with previously treated/metastatic pancreatic cancer. Patients were treated with dinaciclib (6-12 mg/m2 i.v.) and MK-2206 (60-135 mg p.o.) weekly. Tumor biopsies were performed to measure pAKT, pERK, and Ki67 at baseline and after one completed cycle (dose level 2 and beyond). Thirty-nine patients participated in the study. The maximum tolerated doses were dinaciclib 9 mg/m2 and MK-2206 135 mg. Treatment-related grade 3 and 4 toxicities included neutropenia, lymphopenia, anemia, hyperglycemia, hyponatremia, and leukopenia. No objectives responses were observed. Four patients (10%) had stable disease as their best response. At the recommended dose, median survival was 2.2 months. Survival rates at 6 and 12 months were 11% and 5%, respectively. There was a nonsignificant reduction in pAKT composite scores between pretreatment and post-treatment biopsies (mean 0.76 vs. 0.63; P = 0.635). The combination of dinaciclib and MK-2206 was a safe regimen in patients with metastatic pancreatic cancer, although without clinical benefit, possibly due to not attaining biologically effective doses. Given the strong preclinical evidence of Ral and AKT inhibition, further studies with better tolerated agents should be considered.

A Phase I Trial of the VEGF Receptor Tyrosine Kinase Inhibitor Pazopanib in Combination with the MEK Inhibitor Trametinib in Advanced Solid Tumors and Differentiated Thyroid Cancers.

PURPOSE: Differentiated thyroid cancer (DTC) responds to VEGF receptor inhibitors. VEGF signals through RAS/RAF/MEK signaling. We evaluated the safety and efficacy of the VEGF receptor inhibitor pazopanib and MEK inhibitor trametinib in advanced solid tumors and DTC. PATIENTS AND METHODS: Patients with advanced solid tumors were enrolled in a phase I, multicenter trial with a DTC expansion cohort. Patients received pazopanib 400-800 mg and trametinib 1-2 mg daily. Efficacy in the expansion cohort was assessed with objective response (OR) at 6 months of treatment. RESULTS: Twenty-six patients were enrolled in five dose levels. MTD was not reached; the recommended phase II dose was pazopanib 800 mg orally and trametinib 2 mg orally every day. There was one dose-limiting toxicity on dose level 1 with grade 3 fatigue and muscle weakness. Common grade 3 adverse events were elevated transaminases (19%), diarrhea (15%), hypertension (12%), and fatigue (8%). Thirteen patients were enrolled in the DTC cohort; OR was 33% (95% confidence interval, 9.9, 65.1%) and median progression-free survival was 10.7 months. The cohort was terminated after planned interim analysis suggested insufficiently increased activity against the historical control of pazopanib alone. Reduction in tumor diameter negatively correlated with p-ERK change in tumor (Spearman ρ = -0.71; P = 0.05). NRAS mutation was associated with response (Fisher exact P = 0.008). CONCLUSIONS: Pazopanib + trametinib was tolerable at full single-agent doses with clinical activity in DTC but did not achieve the prespecified response rate target.

Cyclin-dependent kinase 5 activity controls cell motility and metastatic potential of prostate cancer cells.

We show here that cyclin-dependent kinase 5 (CDK5), a known regulator of migration in neuronal development, plays an important role in prostate cancer motility and metastasis. P35, an activator of CDK5 that is indicative of its activity, is expressed in a panel of human and rat prostate cancer cell lines, and is also expressed in 87.5% of the human metastatic prostate cancers we examined. Blocking of CDK5 activity with a dominant-negative CDK5 construct, small interfering RNA, or roscovitine resulted in changes in the microtubule cytoskeleton, loss of cellular polarity, and loss of motility. Expression of a dominant-negative CDK5 in the highly metastatic Dunning AT6.3 prostate cancer cell line also greatly impaired invasive capacity. CDK5 activity was important for spontaneous metastasis in vivo; xenografts of AT6.3 cells expressing dominant-negative CDK5 had less than one-fourth the number of lung metastases exhibited by AT6.3 cells expressing the empty vector. These results show that CDK5 activity controls cell motility and metastatic potential in prostate cancer.

O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis.

Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked ß-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.

Selective growth inhibition in BRAF mutant thyroid cancer by the mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244.

CONTEXT: Activating mutations in the BRAF gene, primarily at V600E, are associated with poorer outcomes in patients with papillary thyroid cancer. MAPK kinase (MEK), immediately downstream of BRAF, is a promising target for ras-raf-MEK-ERK pathway inhibition. OBJECTIVE: The objective of the investigation was to study the efficacy of a MEK1/2 inhibitor in thyroid cancer preclinical models with defined BRAF mutation status. EXPERIMENTAL DESIGN: After treatment with the potent MEK 1/2 inhibitor AZD6244, MEK inhibition and cell growth were examined in four BRAF mutant (V600E) and two BRAF wild-type thyroid cancer cell lines and in xenografts from a BRAF mutant cell line. RESULTS: AZD6244 potently inhibited MEK 1/2 activity in thyroid cancer cell lines regardless of BRAF mutation status, as evidenced by reduced ERK phosphorylation. Four BRAF mutant lines exhibited growth inhibition at low doses of the drug, with GI50 concentrations ranging from 14 to 50 nm, predominantly via a G0/G1 arrest, comparable with findings in a sensitive BRAF mutant melanoma cell line. In contrast, two BRAF wild-type lines were significantly less sensitive, with GI50 values greater than 200 nm. Nude mouse xenograft tumors derived from the BRAF mutant line ARO exhibited dose-dependent growth inhibition by AZD6244, with effective treatment at 10 mg/kg by oral gavage. This effect was primarily cytostatic and associated with marked inhibition of ERK phosphorylation. CONCLUSION: AZD6244 inhibits the MEK-ERK pathway across a spectrum of thyroid cancer cells. MEK inhibition is cytostatic in papillary thyroid cancer and anaplastic thyroid cancer cells bearing a BRAF mutation and may have less impact on thyroid cancer cells lacking this mutation.

The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway.

Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.

Notch signaling induces rapid degradation of achaete-scute homolog 1.

In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer.

Recent advances in the biology and therapy of medullary thyroid carcinoma.

Medullary thyroid cancer (MTC) is a relatively uncommon yet prognostically significant thyroid cancer. Several recent advances in the biology and current or potential treatment of MTC are notable. These include a new understanding of the developmental biology of the thyroid C cell, which heretofore was thought to develop from the neural crest. RET, encoded by the most common driver gene in MTC, has been shown to be a dual function kinase, thus expanding its potential substrate repertoire. Promising new therapeutic developments are occurring; many have recently progressed to clinical development. There are new insights into RET inhibitor therapy for MTC. New strategies are being developed to inhibit the RAS proteins, which are potential therapeutic targets in MTC. Potential emerging immunotherapies for MTC are discussed. However, gaps in our knowledge of the basic biology of the C cell, its transformation to MTC, and the mechanisms of resistance to therapy impede progress; further research in these areas would have a substantial impact on the field.

Activity of irinotecan and the tyrosine kinase inhibitor CEP-751 in medullary thyroid cancer.

CONTEXT: Medullary thyroid cancer (MTC) is a cancer of the parafollicular C cells that commonly presents with an inherited or acquired RET gene mutation. There is currently no effective systemic treatment for MTC. OBJECTIVE: The objective of this study was to investigate a systemic therapeutic approach to treat MTC. We studied the sensitivity of an MTC cell line and xenograft to irinotecan, alone and in combination with the tyrosine kinase inhibitor, CEP-751. RESULTS: In TT cell culture and xenografts, irinotecan treatment was highly effective. This effect was augmented by treatment with CEP-751. Treatment of TT cell xenografts resulted in durable complete remission in 100% of the mice, with median time to recurrence of 70 d for irinotecan alone and more than 130 d for irinotecan plus CEP-751. Although irinotecan induced an S phase checkpoint arrest in TT cells, CEP-751 in combination with irinotecan resulted in a loss of this arrest. CEP-751 induced a loss in the induction of the DNA repair program marked by phospho-H2AX and the checkpoint pathway marked by the activated Chk1 pathway. CONCLUSIONS: Irinotecan treatment was highly effective in a preclinical model of human MTC, resulting in complete remission in 100% of the xenografts treated. The duration of remission was further enhanced by combination with the kinase inhibitor, CEP-751. These results suggest that irinotecan, alone or in combination, may be useful for the treatment of MTC.

Epidermal growth factor receptor-independent constitutive activation of STAT3 in head and neck squamous cell carcinoma is mediated by the autocrine/paracrine stimulation of the interleukin 6/gp130 cytokine system.

The aberrant growth of head and neck squamous cell carcinoma (HNSCC) is often associated with the constitutive activation of signal-transducer-and-activator-of-transcription-3 (STAT3), which is believed to result from the persistent stimulation of EGF receptors that are highly expressed in squamous cell carcinoma (SCC) cells. To investigate the mechanism underlying STAT3 deregulation in HNSCC, we examined the interplay of the STAT3 and epidermal growth factor receptor (EGFR) signaling pathways using a panel of HNSCC cell lines. Although STAT3 was active in most HNSCC cell lines, only 3 of 10 HNSCC cell lines were moderately to strongly positive for activated EGFR. Even in the EGFR-positive cell lines, STAT3 activation was not dependent on EGFR activation, as STAT3 tyrosine phosphorylation levels persisted after treatment with AG1478, a chemical inhibitor of EGFR activity. Furthermore, we found that conditioned medium harvested from HNSCC cells could induce STAT3 tyrosine phosphorylation in immortalized keratinocytes regardless of the status of EGFR signaling. In contrast, blocking the cytokine gp130 coreceptor abolished STAT3 tyrosine phosphorylation in HNSCC cells and that induced by the conditioned medium. Immunodepletion studies suggested interleukin 6 (IL6) as the major autocrine/paracrine factor for STAT3 activation, which coincided with high levels of secretion of IL6 into the culture medium by these cancer cells. Treatment with a specific inhibitor of Janus kinase, AG490, in HNSCC cells led to a reduction of active STAT3 and caused significant growth retardation and apoptosis. Thus, constitutive activation of STAT3 in HNSCC may use an autocrine/paracrine-activating loop mediated by IL6 and other cytokines acting through the gp130 receptor family, which may confer both proliferative and survival potential in this malignancy.

CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth.

All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations <100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression.

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Regulation of neuroendocrine differentiation in gastrointestinal carcinoid tumor cells by notch signaling.

CONTEXT: Gastrointestinal (GI) carcinoid tumors elaborate serotonin and other vasoactive substances, causing the carcinoid syndrome. Based on developmental biology data, we hypothesized that basic helix-loop-helix transcription factors, including achaete-scute complex homolog-like 1 (Ascl1)/hASH1, and the Notch signaling pathway might regulate the neuroendocrine phenotype in GI carcinoids. OBJECTIVE: The aim of this study was to evaluate expression of developmental transcription factors and Notch signaling components in GI carcinoids and model their interaction in a relevant GI carcinoid cell line. DESIGN: Fourteen GI carcinoid tumor specimens, five paired adjacent normal tissues, fetal tissues, and tumor cell lines were analyzed by RT-PCR and immunoblot. BON carcinoid cells were further analyzed after Notch overexpression for neuroendocrine marker expression, serotonin production, and growth. SETTING: The study was conducted in an academic referral center. PATIENTS OR OTHER PARTICIPANTS: Deidentified archival pathology specimens were examined. RESULTS: Among a panel of six developmental transcription factors tested, only Ascl1 mRNA was overexpressed compared with surrounding normal tissue (seven of 10 GI carcinoid tumors and in BON cells, none of five normal tissues). Ascl1 protein was also expressed in four of four carcinoid tumors and BON cells). Notch pathway ligands, receptors, and downstream effectors were widely expressed in tumor and normal specimens. Overexpression of activated Notch1 in BON cells led to induction of the Notch effector hairy and enhancer of split 1 (Hes1), loss of Ascl1, reductions in neuron-specific enolase, synaptophysin, and chromogranin A, and most significantly, an 89% decrease in serotonin concentration and equivalent reductions in serotonin-reactive cells and repression of tryptophan hydroxylase 1 mRNA. CONCLUSIONS: The Notch signaling pathway is a significant regulator of neuroendocrine differentiation and serotonin production in GI carcinoid tumors.

Trametinib with and without pazopanib has potent preclinical activity in thyroid cancer.

Multikinase inhibitors (MKIs) targeting VEGF receptors and other receptor tyrosine kinases have shown considerable activity in clinical trials of thyroid cancer. Thyroid cancer frequently exhibits activation of the RAS/RAF/MEK/ERK pathway. In other types of cancer, paradoxical ERK activation has emerged as a potential resistance mechanism to RAF-inhibiting drugs including MKIs such as sorafenib and pazopanib. We therefore queried whether the MEK inhibitor trametinib, could augment the activity of pazopanib in thyroid cancer cell lines. Trametinib potently inhibited growth in vitro (GI50 1.1-4.8 nM), whereas pazopanib had more limited in vitro activity, as anticipated (GI50 1.4-7.1 µM). We observed progressive upregulation of ERK activity with pazopanib treatment, an effect abrogated by trametinib. For xenografts (bearing either KRASG12R or BRAFV600E mutations), the combination of trametinib and pazopanib led to sustained shrinkage in tumor volume by 50% or more, compared to pre-treatment baseline. Trametinib also was highly effective as a single agent, compared to pazopanib alone. These preclinical findings support the evaluation of trametinib, alone or in combination with pazopanib or other kinase inhibitors, in thyroid cancer clinical trials. We highlight the importance of pharmacodynamic assessment of the ERK pathway for patients enrolled in trials involving MKIs.

Combined Inhibition of Cyclin-Dependent Kinases (Dinaciclib) and AKT (MK-2206) Blocks Pancreatic Tumor Growth and Metastases in Patient-Derived Xenograft Models.

KRAS is activated by mutation in the vast majority of cases of pancreatic cancer; unfortunately, therapeutic attempts to inhibit KRAS directly have been unsuccessful. Our previous studies showed that inhibition of cyclin-dependent kinase 5 (CDK5) reduces pancreatic cancer growth and progression, through blockage of the centrally important RAL effector pathway, downstream of KRAS. In the current study, the therapeutic effects of combining the CDK inhibitor dinaciclib (SCH727965; MK-7965) with the pan-AKT inhibitor MK-2206 were evaluated using orthotopic and subcutaneous patient-derived human pancreatic cancer xenograft models. The combination of dinaciclib (20 mg/kg, i.p., three times a week) and MK-2206 (60 mg/kg, orally, three times a week) dramatically blocked tumor growth and metastasis in all eight pancreatic cancer models examined. Remarkably, several complete responses were induced by the combination treatment of dinaciclib and MK-2206. The striking results obtained in these models demonstrate that the combination of dinaciclib with the pan-AKT inhibitor MK-2206 is promising for therapeutic evaluation in pancreatic cancer, and strongly suggest that blocking RAL in combination with other effector pathways downstream from KRAS may provide increased efficacy in pancreatic cancer. Based on these data, an NCI-CTEP-approved multicenter phase I clinical trial for pancreatic cancer of the combination of dinaciclib and MK-2206 (NCT01783171) has now been opened.

The homeobox gene BARX2 can modulate cisplatin sensitivity in human epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most common cause of death from gynaecological malignancy. Resistance to platinum chemotherapy is a major reason for treatment failure and poor prognosis. The human homeobox gene BARX2 is located within a minimal region at 11q25 that is associated with frequent loss of heterozygosity (LOH) and adverse survival in EOC. BARX2 is a transcription factor known to regulate transcription of specific cell adhesion molecules in the mouse. We have previously shown that BARX2 expression is low in clear cell/endometrioid and high in serous adenocarcinomas of the ovary, histologic variants that are less and more sensitive, respectively to platinum chemotherapy. The aim of this study was to define whether BARX2 could modulate sensitivity to cisplatin in human epithelial ovarian cancer. In two cell line series sequentially derived from ovarian cancer patients pre- and post-cisplatin chemotherapy, BARX2 expression was downregulated in the cell lines established upon tumor recurrence after platinum therapy. Transfection of BARX2 into a platinum resistant cell line significantly reversed cisplatin resistance compared with its isogenic platinum sensitive parent, in both growth inhibition and clonogenic assays. Taken together, our data demonstrate that the homeobox gene BARX2 may be a biological factor involved in determining sensitivity or resistance to the cytotoxic effects of cisplatin.

Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression.

Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice.

Cabozantinib in progressive medullary thyroid cancer.

PURPOSE: Cabozantinib, a tyrosine kinase inhibitor (TKI) of hepatocyte growth factor receptor (MET), vascular endothelial growth factor receptor 2, and rearranged during transfection (RET), demonstrated clinical activity in patients with medullary thyroid cancer (MTC) in phase I. PATIENTS AND METHODS: We conducted a double-blind, phase III trial comparing cabozantinib with placebo in 330 patients with documented radiographic progression of metastatic MTC. Patients were randomly assigned (2:1) to cabozantinib (140 mg per day) or placebo. The primary end point was progression-free survival (PFS). Additional outcome measures included tumor response rate, overall survival, and safety. RESULTS: The estimated median PFS was 11.2 months for cabozantinib versus 4.0 months for placebo (hazard ratio, 0.28; 95% CI, 0.19 to 0.40; P < .001). Prolonged PFS with cabozantinib was observed across all subgroups including by age, prior TKI treatment, and RET mutation status (hereditary or sporadic). Response rate was 28% for cabozantinib and 0% for placebo; responses were seen regardless of RET mutation status. Kaplan-Meier estimates of patients alive and progression-free at 1 year are 47.3% for cabozantinib and 7.2% for placebo. Common cabozantinib-associated adverse events included diarrhea, palmar-plantar erythrodysesthesia, decreased weight and appetite, nausea, and fatigue and resulted in dose reductions in 79% and holds in 65% of patients. Adverse events led to treatment discontinuation in 16% of cabozantinib-treated patients and in 8% of placebo-treated patients. CONCLUSION: Cabozantinib (140 mg per day) achieved a statistically significant improvement of PFS in patients with progressive metastatic MTC and represents an important new treatment option for patients with this rare disease. This dose of cabozantinib was associated with significant but manageable toxicity.