Multiple pathways coordinating reprogramming of endothelial cells into osteoblasts by BMP4.

Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3ß-ßcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and ß-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that ß-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.

Radium-223 Treatment Increases Immune Checkpoint Expression in Extracellular Vesicles from the Metastatic Prostate Cancer Bone Microenvironment.

PURPOSE: Radium-223 prolongs survival in a fraction of men with bone metastatic prostate cancer (PCa). However, there are no markers for monitoring response and resistance to Radium-223 treatment. Exosomes are mediators of intercellular communication and may reflect response of the bone microenvironment to Radium-223 treatment. We performed molecular profiling of exosomes and compared the molecular profile in patients with favorable and unfavorable overall survival. EXPERIMENTAL DESIGN: We performed exosomal transcriptome analysis in plasma derived from our preclinical models (MDA-PCa 118b tumors, TRAMP-C2/BMP4 PCa) and from the plasma of 25 patients (paired baseline and end of treatment) treated with Radium-223. All samples were run in duplicate, and array data analyzed with fold changes +2 to -2 and P < 0.05. RESULTS: We utilized the preclinical models to establish that genes derived from the tumor and the tumor-associated bone microenvironment (bTME) are differentially enriched in plasma exosomes upon Radium-223 treatment. The mouse transcriptome analysis revealed changes in bone-related and DNA damage repair-related pathways. Similar findings were observed in plasma-derived exosomes from patients treated with Radium-223 detected changes. In addition, exosomal transcripts detected immune-suppressors (e.g., PD-L1) that were associated with shorter survival to Radium-223. Treatment of the Myc-CaP mouse model with a combination of Radium-223 and immune checkpoint therapy (ICT) resulted in greater efficacy than monotherapy. CONCLUSIONS: These clinical and coclinical analyses showed that RNA profiling of plasma exosomes may be used for monitoring the bTME in response to treatment and that ICT may be used to increase the efficacy of Radium-223.

Cabozantinib Reverses Renal Cell Carcinoma-mediated Osteoblast Inhibition in Three-dimensional Coculture In Vitro and Reduces Bone Osteolysis In Vivo.

Renal cell carcinoma bone metastases (RCCBM) are typically osteolytic. We previously showed that BIGH3 (beta Ig-h3/TGFBI), secreted by 786-O renal cell carcinoma, plays a role in osteolytic bone lesion in RCCBM through inhibition of osteoblast (OSB) differentiation. To study this interaction, we employed three-dimensional (3D) hydrogels to coculture bone-derived 786-O (Bo-786) renal cell carcinoma cells with MC3T3-E1 pre-OSBs. Culturing pre-OSBs in the 3D hydrogels preserved their ability to differentiate into mature OSB; however, this process was decreased when pre-OSBs were cocultured with Bo-786 cells. Knockdown of BIGH3 in Bo-786 cells recovered OSB differentiation. Furthermore, treatment with bone morphogenetic protein 4, which stimulates OSB differentiation, or cabozantinib (CBZ), which inhibits VEGFR1 and MET tyrosine kinase activities, also increased OSB differentiation in the coculture. CBZ also inhibited pre-osteoclast RAW264.7 cell differentiation. Using RCCBM mouse models, we showed that CBZ inhibited Bo-786 tumor growth in bone. CBZ treatment also increased bone volume and OSB number, and decreased osteoclast number and blood vessel density. When tested in SN12PM6 renal cell carcinoma cells that have been transduced to overexpress BIGH3, CBZ also inhibited SN12PM6 tumor growth in bone. These observations suggest that enhancing OSB differentiation could be one of the therapeutic strategies for treating RCCBM that exhibit OSB inhibition characteristics, and that this 3D coculture system is an effective tool for screening osteoanabolic agents for further in vivo studies.

A Phase II Study of Cabozantinib and Androgen Ablation in Patients with Hormone-Naïve Metastatic Prostate Cancer.

PURPOSE: Cabozantinib, an oral inhibitor of c-MET/VEGFR2 signaling, improved progression-free survival (mPFS) but not overall survival (OS) in metastatic castrate-resistant prostate cancer. We evaluated cabozantinib plus androgen deprivation therapy (ADT) in hormone-naïve metastatic prostate cancer (HNMPCa). PATIENTS AND METHODS: Patients received ADT plus cabozantinib starting at 60 mg daily. The primary endpoint was castrate-resistant PFS by radiographic criteria, clinical progression, or receipt of additional therapy. Secondary endpoints included OS, safety, radiographic responses, and biomarker modulation. RESULTS: Sixty-two patients received treatment. With a median follow-up of 31.2 months, the mPFS was 16.1 months (95% CI, 14.6-22.7 months), and mOS was not reached. Reductions in PSA ≥ 90%, bone-specific alkaline phosphatase ≥ 50%, and urine N-telopeptides ≥ 50% occurred in 83%, 87%, and 86% of evaluable patients, respectively. Responses in bone scan and measurable disease were observed in 81% of and 90% of evaluable patients, respectively. Most common grade 3 adverse events were hypertension (19%), diarrhea (6%), and thromboembolic events (6%), and dose reductions occurred in 85% of patients. Analysis of baseline cytokine and angiogenic factors (CAFs) revealed that higher plasma concentrations of Lumican, CXCL5, CD25, and CD30 were associated with shorter PFS as was high tumor expression of pFGFR1. CONCLUSIONS: Cabozantinib plus ADT has promising clinical activity in HNMPCa. CAF profiles and tissue markers suggest candidate prognostic and predictive markers of cabozantinib benefit and provide insights for rational therapy combinations.

Potentially inappropriate medications defined by STOPP criteria in older patients with breast and colorectal cancer.

PURPOSE: Screening for potentially inappropriate medications (PIM) is recommended in older patients with cancer receiving chemotherapy, given the concern for adverse drug reactions, drug-drug interactions and non-adherence. Our objective was to determine the impact of PIM on outcomes in patients with breast and colorectal cancers receiving chemotherapy. METHODS: We used data from the SEER-Medicare database, including patients >/= 66 years old with a diagnosis of Stage II/III breast and colorectal cancer made between 7/1/2007-12/31/2009. We used modified STOPP criteria to define baseline PIM as a dichotomous variable in the 4 months prior to diagnosis. STOPP criteria was used based on its performance as a robust measure of PIM. Outcomes measures included ER visits, hospitalizations, and death within 3 months from the last chemotherapy, and a composite of the three. We used Chi-square or Fisher's exact test to determine associations of PIM with covariates and outcomes, and Cox proportional hazards (PH) model for the time-to-event analysis. RESULTS: Final analysis included 1,595 patients with breast cancer and 1,528 patients with colorectal cancer. Frequency of baseline PIM by STOPP was 31.5% in the breast and 30.9% in the colorectal cohort. In the breast cohort, associations with the composite outcome in the Cox PH model included disease stage, comorbidity, medication number and baseline ER visits/hospitalization. Age, gender, race, comorbidity and baseline ER visits/hospitalization were associated in the colorectal cohort. PIM was not associated with outcomes in either cohort, aside from hospitalization in the breast. CONCLUSIONS: We found no consistent association between pre-chemotherapy PIM defined by STOPP and outcomes.

Potentially inappropriate medication use in older patients with breast and colorectal cancer.

BACKGROUND: The objective of this study was to determine patient characteristics associated with potentially inappropriate medication (PIM) use and its impact on outcomes for patients with breast or colorectal cancer receiving adjuvant chemotherapy. METHODS: The Surveillance, Epidemiology, and End Results database, linked to Medicare claims, was used. The cohort included patients who were 66 years old or older and were diagnosed with stage II or III breast or colorectal cancer between July 1, 2007, and December 31, 2009. The Drugs to Avoid in the Elderly (DAE) list and the Beers criteria were used to identify PIM use. Univariate/multivariate logistic regression determined the association of baseline PIMs with covariates. Event-free survival (EFS) was defined as the time from chemotherapy initiation to the first emergency room (ER) visit, hospitalization, death, or a composite until 3 months after chemotherapy. Cox proportional hazards modeling determined the association of PIMs with EFS. RESULTS: The analysis included 1595 patients with breast cancer and 1528 patients with colorectal cancer. The baseline PIM frequencies were 22.2% (according to the DAE list) and 27.6% (according to the Beers criteria) in the breast cohort and 15.5% (according to the DAE list) and 24.8% (according to the Beers criteria) in the colorectal cohort. Among patients with breast cancer, 37.5% had at least 1 adverse outcome; associations included the use of ≥5 medications, an advanced stage, higher comorbidity, and prior ER visits/hospitalizations. Baseline PIM use according to the DAE list was associated with an increased risk of death in patients with breast cancer. Among patients with colorectal cancer, 45% had at least 1 adverse outcome, and associations included the use of ≥5 medications, older age, female sex, and higher comorbidity. A time-to-event analysis revealed no association between baseline PIM use and most outcomes. CONCLUSIONS: These findings require further prospective confirmation, but they support a correlation between polypharmacy and adverse outcomes for cancer patients and call into question the association with PIMs. Cancer 2018;124:3000-7. © 2018 American Cancer Society.

Osteoblast-Secreted Factors Mediate Dormancy of Metastatic Prostate Cancer in the Bone via Activation of the TGFßRIII-p38MAPK-pS249/T252RB Pathway.

Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFß2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFß2 and GDF10 induced dormancy through TGFßRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both in vitro and in vivo Lower TGFßRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFßRIII-p38MAPK-pS249/pT252-RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone.Significance: These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Cancer Res; 78(11); 2911-24. ©2018 AACR.

Organelle-Derived Acetyl-CoA Promotes Prostate Cancer Cell Survival, Migration, and Metastasis via Activation of Calmodulin Kinase II.

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the ß-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle ß-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to ß-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.

BIGH3 Promotes Osteolytic Lesions in Renal Cell Carcinoma Bone Metastasis by Inhibiting Osteoblast Differentiation.

BACKGROUND: Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. METHODS: We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. RESULTS: We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens. CONCLUSIONS: These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.

Cabozantinib-induced osteoblast secretome promotes survival and migration of metastatic prostate cancer cells in bone.

Therapies that target cancer cells may have unexpected effects on the tumor microenvironment that affects therapy outcomes or render therapy resistance. Prostate cancer (PCa) bone metastasis is uniquely associated with osteoblastic bone lesions and treatment with cabozantinib, a VEGFR-2 and MET inhibitor, leads to a reduction in number and/or intensity of lesions on bone scans. However, resistance to cabozantinib therapy inevitably occurs. We examined the effect of cabozantinib on osteoblast differentiation and secretion in the context of therapy resistance. We showed that primary mouse osteoblasts express VEGFR2 and MET and cabozantinib treatment decreased osteoblast proliferation but enhanced their differentiation. A genome-wide analysis of transcriptional responses of osteoblasts to cabozantinib identified a set of genes accounting for inhibition of proliferation and stimulation of differentiation, and a spectrum of secreted proteins induced by cabozantinib, including pappalysin, IGFBP2, WNT 16, and DKK1. We determined that these proteins were upregulated in the conditioned medium of cabozantinib-treated osteoblasts (CBZ-CM) compared to control CM. Treatment of C4-2B4 or PC3-mm2 PCa cells with CBZ-CM increased the anchorage-independent growth and migration of these PCa cells compared to cells treated with control CM. These results suggest that the effect of cabozantinib on the tumor microenvironment may increase tumor cell survival and cause therapy resistance.

JAK2-binding long noncoding RNA promotes breast cancer brain metastasis.

Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M- and IL-6-triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease.

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells.

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130-140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer.

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2cre/Osxf/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osxf/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

Androgen receptor inhibitor-induced "BRCAness" and PARP inhibition are synthetically lethal for castration-resistant prostate cancer.

Cancers with loss-of-function mutations in BRCA1 or BRCA2 are deficient in the DNA damage repair pathway called homologous recombination (HR), rendering these cancers exquisitely vulnerable to poly(ADP-ribose) polymerase (PARP) inhibitors. This functional state and therapeutic sensitivity is referred to as "BRCAness" and is most commonly associated with some breast cancer types. Pharmaceutical induction of BRCAness could expand the use of PARP inhibitors to other tumor types. For example, BRCA mutations are present in only ~20% of prostate cancer patients. We found that castration-resistant prostate cancer (CRPC) cells showed increased expression of a set of HR-associated genes, including BRCA1, RAD54L, and RMI2 Although androgen-targeted therapy is typically not effective in CRPC patients, the androgen receptor inhibitor enzalutamide suppressed the expression of those HR genes in CRPC cells, thus creating HR deficiency and BRCAness. A "lead-in" treatment strategy, in which enzalutamide was followed by the PARP inhibitor olaparib, promoted DNA damage-induced cell death and inhibited clonal proliferation of prostate cancer cells in culture and suppressed the growth of prostate cancer xenografts in mice. Thus, antiandrogen and PARP inhibitor combination therapy may be effective for CRPC patients and suggests that pharmaceutically inducing BRCAness may expand the clinical use of PARP inhibitors.

Dual Inhibition of EGFR and c-Src by Cetuximab and Dasatinib Combined with FOLFOX Chemotherapy in Patients with Metastatic Colorectal Cancer.

Purpose: Aberrant activation of the intracellular tyrosine kinase Src has been implicated as a mechanism of acquired chemotherapy resistance in metastatic colorectal cancer (mCRC). Here, the oral tyrosine kinase Src inhibitor, dasatinib, was investigated in combination with FOLFOX and cetuximab.Experimental Design: We performed a phase IB/II study of 77 patients with previously treated mCRC. Primary objectives were to determine the maximum tolerated dose, dose-limiting toxicities (DLT), pharmacodynamics, and efficacy. Using a 3 + 3 design, patients received FOLFOX6 with cetuximab and escalating doses of dasatinib (100, 150, 200 mg daily), followed by a 12-patient expansion cohort at 150 mg. Phase II studies evaluated FOLFOX plus dasatinib 100 mg in KRAS c12/13mut patients or in combination with cetuximab if KRAS c12/13WT FAK and paxillin were utilized as surrogate blood biomarkers of Src inhibition, and paired biopsies of liver metastases were obtained in patients in the expansion cohort.Results: In phase IB, the DLTs were grade 3/4 fatigue (20%) and neutropenia (23%). In phase II, grade 3/4 fatigue (23%) and pleural effusions (11%) were present. Response rates were 20% (6 of 30) in the phase IB escalation and expansion cohort and 13% (3 of 24) and 0% (0 of 23) in the KRAS c12/13WT and mutant cohorts of phase II, respectively. Median progression-free survival was 4.6, 2.3, and 2.3 months, respectively. There was no evidence of Src inhibition based on surrogate blood biomarkers or paired tumor biopsies.Conclusions: The combination of dasatinib plus FOLFOX with or without cetuximab showed only modest clinical activity in refractory colorectal cancer. This appears to be primarily due to a failure to fully inhibit Src at the achievable doses of dasatinib. The combination of dasatinib plus FOLFOX with or without cetuximab did not show meaningful clinical activity in refractory colorectal cancer due to failure to fully inhibit Src. Clin Cancer Res; 23(15); 4146-54. ©2017 AACR.

Targeting DNA Damage Response in Prostate Cancer by Inhibiting Androgen Receptor-CDC6-ATR-Chk1 Signaling.

Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.

A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis.

Bone metastases remain a serious health concern because of limited therapeutic options. Here, we report that crosstalk between ROR1-HER3 and the Hippo-YAP pathway promotes breast cancer bone metastasis in a long noncoding RNA-dependent fashion. Mechanistically, the orphan receptor tyrosine kinase ROR1 phosphorylates HER3 at a previously unidentified site Tyr1307, following neuregulin stimulation, independently of other ErbB family members. p-HER3 Tyr1307 recruits the LLGL2-MAYA-NSUN6 RNA-protein complex to methylate Hippo/MST1 at Lys59. This methylation leads to MST1 inactivation and activation of YAP target genes in tumour cells, which elicits osteoclast differentiation and bone metastasis. Furthermore, increased ROR1, p-HER3 Tyr1307 and MAYA levels correlate with tumour metastasis and unfavourable outcomes. Our data provide insights into the mechanistic regulation and linkage of the ROR1-HER3 and Hippo-YAP pathway in a cancer-specific context, and also imply valuable therapeutic targets for bone metastasis and possible therapy-resistant tumours.

The combination of serum insulin, osteopontin, and hepatocyte growth factor predicts time to castration-resistant progression in androgen dependent metastatic prostate cancer- an exploratory study.

BACKGROUND: We hypothesized that pretreatment serum levels of insulin and other serum markers would predict Progression-free survival (PFS), defined as time to castration-resistant progression or death, in metastatic androgen-dependent prostate cancer (mADPC). METHODS: Serum samples from treatment-naïve men participating in a randomized phase 3 trial of ADT +/- chemotherapy were retrospectively analyzed using multiplex assays for insulin and multiple other soluble factors. Cox proportional hazards regression models were used to identify associations between individual factor levels and PFS. RESULTS: Sixty six patients were evaluable (median age = 72 years; median prostate surface antigen [PSA] = 31.5 ng/mL; Caucasian = 86 %; Gleason score ≥8 = 77 %). In the univariable analysis, higher insulin (HR = 0.81 [0.67, 0.98] p = 0.03) and C-peptide (HR = 0.62 [0.39, 1.00]; p = 0.05) levels were associated with a longer PFS, while higher Hepatocyte Growth Factor (HGF; HR = 1.63 [1.06, 2.51] p = 0.03) and Osteopontin (OPN; HR = 1.56 [1.13, 2.15]; p = 0.01) levels were associated with a shorter PFS. In multivariable analysis, insulin below 2.1 (ln scale; HR = 2.55 [1.24, 5.23]; p = 0.011) and HGF above 8.9 (ln scale; HR = 2.67 [1.08, 3.70]; p = 0.027) levels were associated with longer PFS, while adjusted by OPN, C-peptide, trial therapy and metastatic volume. Four distinct risk groups were identified by counting the number of risk factors (RF) including low insulin, high HGF, high OPN levels, and low C-peptide levels (0, 1, 2, and 3). Median PFS was 9.8, 2.0, 1.6, and 0.7 years for each, respectively (p < 0.001). CONCLUSION: Pretreatment serum insulin, HGF, OPN, and C-peptide levels can predict PFS in men with mADPC treated with ADT. Risk groups based on these factors are superior predictors of PFS than each marker alone.