Joseph L. Sottnik

Double-Blind, Randomized, Phase 2 Trial of Maintenance Sunitinib Versus Placebo After Response to Chemotherapy in Patients With Advanced Urothelial Carcinoma

Angiogenesis contributes to the progression of urothelial carcinoma (UC). In the current study, the authors investigated the role of maintenance sunitinib in patients with advanced UC.

Tumor-induced pressure in the bone microenvironment causes osteocytes to promote the growth of prostate cancer bone metastases.

Cross-talk between tumor cells and their microenvironment is critical for malignant progression. Cross-talk mediators, including soluble factors and direct cell contact, have been identified, but roles for the interaction of physical forces between tumor cells and the bone microenvironment have not been described. Here, we report preclinical evidence that tumor-generated pressure acts to modify the bone microenvironment to promote the growth of prostate cancer bone metastases. Tumors growing in mouse tibiae increased intraosseous pressure. Application of pressure to osteocytes, the main mechanotransducing cells in bone, induced prostate cancer growth and invasion. Mechanistic investigations revealed that this process was mediated in part by upregulation of CCL5 and matrix metalloproteinases in osteocytes. Our results defined the critical contribution of physical forces to tumor cell growth in the tumor microenvironment, and they identified osteocytes as a critical mediator in the bone metastatic niche.

Integrin alpha2beta1 (α2β1) promotes prostate cancer skeletal metastasis

Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin α2β1 stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we sought to determine whether α2β1 integrin is a potential mediator of skeletal metastasis. To examine whether α2β1 integrin mediates PCa metastasis, α2 integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaPcol PCa cells. We document that the over-expression of α2 cDNA stimulated whereas α2 shRNA inhibited the ability of transduced cells to bind to or migrate towards collagen in vitro. Correspondingly, α2 integrin knock-down reduced the tumor burden of intra-osseous tumors compared to control-transduced cells. To investigate the clinical significance of α2β1 expression in PCa, α2β1 protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that α2β1 protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that α2β1 contributes to the selective metastasis to the bone. Taken together, these data support that α2β1 integrin is needed for the efficient metastasis of PCa cells to the skeleton.

Understanding and targeting osteoclastic activity in prostate cancer bone metastases

Bone metastasis is a debilitating side effect of advanced prostatic carcinoma impacting nearly all of the men developing this disease. Even though a majority of these lesions are considered osteoblastic, it is believed that there is an underlying osteolytic component. Lytic processes are governed primarily by osteoclasts, the primary bone resorptive cell. Osteolysis has been implicated in tumor cell seeding and nourishment of tumor growth via development of pro-tumorigenic changes in the microenvironment. Herein, we provide a current view of the processes involved in regulating osteolysis in the presence of prostate cancer bone metastases. Several factors have been implicated in the division, differentiation, and activation of osteoclasts, including, but not limited to, interleukin-6, receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and parathyroid hormone-related protein (PTHrP). Effector molecules in bone resorption play a significant role, such as matrix metalloproteinases (MMPs), cathepsins, and acid secretion. The primary method for treating skeletal events associated with prostate cancer bone metastases has been bisphosphonates. However, a new therapeutic, denosumab, a monoclonal antibody that inhibits RANKL in a mechanism similar to that attributed to the endogenous mediator OPG, has received approval for treatment of skeletally associated metastases. Additional novel targets are continuously being developed for bone metastases. In this review, we describe the processes involved in osteolysis of the prostate cancer bone microenvironment, and introduce therapeutics that may play a role in inhibiting tumor growth leading to increased survival and quality of life.

The PCa Tumor Microenvironment.

The tumor microenvironment (TME) is a very complex niche that consists of multiple cell types, supportive matrix and soluble factors. Cells in the TME consist of both host cells that are present at tumor site at the onset of tumor growth and cells that are recruited in either response to tumor- or host-derived factors. PCa (PCa) thrives on crosstalk between tumor cells and the TME. Crosstalk results in an orchestrated evolution of both the tumor and microenvironment as the tumor progresses. The TME reacts to PCa-produced soluble factors as well as direct interaction with PCa cells. In return, the TME produces soluble factors, structural support and direct contact interactions that influence the establishment and progression of PCa. In this review, we focus on the host side of the equation to provide a foundation for understanding how different aspects of the TME contribute to PCa progression. We discuss immune effector cells, specialized niches, such as the vascular and bone marrow, and several key protein factors that mediate host effects on PCa. This discussion highlights the concept that the TME offers a potentially very fertile target for PCa therapy.

Wnt and Wnt inhibitors in bone metastasis

Bone metastasis is a clinically devastating development of progressive cancers including prostate carcinoma, breast carcinoma and multiple myeloma. Bone metastases are typically painful, lead to adverse skeletal-related events, such as fracture, and are highly resistant to therapy. A major contribution to the ability of cancers to successfully establish bone metastases is their ability to exploit mechanisms of normal bone remodeling. Wnts are a large family of morphogenic proteins that are critical for bone development and contribute to maintaining bone mass in the mature organism. Wnt function is balanced by the presence of a variety of endogenous inhibitors, such as the dickkopf family members, secreted frizzled related proteins and sclerostin. Together, these factors contribute to normal bone homeostasis, allowing for dynamic changes in bone to withstand alterations in physical forces and physiological needs. In this review, we describe the role that Wnts and their inhibitors have in normal bone biology and cancer-related bone pathology. An overview of Wnt signaling pathways is discussed and key bone microenvironment cellular players, as they pertain to Wnt biology, are examined. Finally, we describe clinical trials of several Wnt inhibitor antagonists for patients with tumor-related bone disease. As few options currently exist for the treatment of bone-metastatic disease, Wnt proteins and their inhibitors offer promise for the development of novel therapeutics.

Parathyroid hormone-related protein inhibits DKK1 expression through c-Jun-mediated inhibition of β-catenin activation of the DKK1 promoter in prostate cancer.

Prostate cancer (PCa)bone metastases are unique in that majority of them induce excessive mineralized bone matrix, through undefined mechanisms, as opposed to most other cancers that induce bone resorption. Parathyroid hormone-related protein (PTHrP) is produced by PCa cells and intermittent PTHrP exposure has bone anabolic effects, suggesting that PTHrP could contribute to the excess bone mineralization. Wnts are bone-productive factors produced by PCa cells, and the Wnt inhibitor Dickkopfs-1 (DKK1) has been shown to promote PCa progression. These findings, in conjunction with the observation that PTHrP expression increases and DKK1 expression decreases as PCa progresses, led to the hypothesis that PTHrP could be a negative regulator of DKK1 expression in PCa cells and, hence, allow the osteoblastic activity of Wnts to be realized. To test this, we first demonstrated that PTHrP downregulated DKK1 mRNA and protein expression. We then found through multiple mutated DKK1 promoter assays that PTHrP, through c-Jun activation, downregulated the DKK1 promoter through a transcription factor (TCF) response element site. Furthermore, chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that PTHrP mediated this effect through inducing c-Jun to bind to a transcriptional activator complex consisting of β-catenin, which binds the most proximal DKK1 promoter, the TCF response element. Together, these results demonstrate a novel signaling linkage between PTHrP and Wnt signaling pathways that results in downregulation of a Wnt inhibitor allowing for Wnt activity that could contribute the osteoblastic nature of PCa.

Osteocytes serve as a progenitor cell of osteosarcoma

Osteosarcoma (OSA) is the most common primary bone tumor in humans. However, the cell of origin of OSA is not clearly defined although there is evidence that osteoblasts may serve as OSA progenitors. The role of osteocytes, terminally differentiated osteoblasts, as OSA progenitors has yet to be described. Analysis of patient cDNA from publicly available microarray data revealed that patients with OSA have increased expression of dentin matrix phosphoprotein 1 (DMP1), a marker of osteocytes. Analysis of multiple murine, human, and canine OSA cell lines revealed DMP1 expression. To test the tumorigenic potential of osteocytes, MLO‐Y4, a SV‐40 immortalized murine osteocyte cell line, was injected into subcutaneous and orthotopic (intratibial) sites of mice. Tumor growth occurred in both locations. Orthotopic MLO‐Y4 tumors produced mixed osteoblastic/osteolytic radiographic lesions; a hallmark of OSA. Together, these data demonstrate for the first time that osteocytes can serve as OSA progenitors. J. Cell. Biochem. 115: 1420–1429, 2014.

Randomized phase II trial of maintenance sunitinib versus placebo following response to chemotherapy (CT) for patients (pts) with advanced urothelial carcinoma (UC).

265 Background: UC response to CT is not durable. Angiogenesis may play a role in the progression of UC. We evaluated whether maintenance sunitinib delays progression after response to CT. METHODS Pts with ECOG PS 0-2, adequate organ function, stable disease, partial or complete response (SD, PR, CR) after 4-6 cycles of CT for advanced UC were randomized to oral sunitinib 50 mg/day, 28 days on, 14 days off (6-week cycle) or placebo. Disease was assessed every 12 wks. At progression, placebo pts were offered open label sunitinib. Primary endpoint: progression rate at 6 months (ms); secondary endpoints: safety, objective response rate, survival, VEGF/sVEGFR2 serum level changes. Using a randomized selection design 42 pts/arm has 90% probability of selecting sunitinib if true reduction in 6-ms progression rate is 15% (from 50% to 35%). RESULTS Study was closed early due to slow accrual. 54 pts with median age 69 years, median ECOG PS 1, 70% with bladder primary, 26 with SD, 23 with PR, 5 with CR to CT were randomized to sunitinib (26) or placebo (28). The median number of cycles was 2/arm (sunitinib 0-15, placebo 0-13). The 6-ms progression rate was 81% (95%CI 61-93%), median time-to-progression (TTP) 5 ms (0.3-22.2, 95%CI 2.4-6.3) for sunitinib and 75% (95%CI 55-89%), 2.7 ms (0.8-19.6, 95%CI 2.5-7.4) for placebo. Response rate in pts with SD at enrollment was 9% for sunitinib and 7% for placebo. Most common G3/4 AEs on sunitinib were diarrhea (15.4%/0%), fatigue (15.4%/3.8%), thrombocytopenia (15.4%/7.7%), hypertension (11.5%/0%). 16 placebo pts received sunitinib with best response 1 PR (6.25%), 6 SD (37.5%), 5 PD (31.25%); 4 not response evaluable. Median TTP was 3.4 ms (0.1-22, 95%CI 1.6-5.5). 11 pts had G3/4 AEs, 5 pts discontinued sunitinib due to AEs (4 related, 1 unrelated). Placebo pts had no change in VEGF/sVEGFR2 over time. Sunitinib pts had no change in VEGF but sVEGFR2 significantly decreased after 1 cycle (p<0.0001) and at progression (p=0.0002). VEGF/sVEGFR2 did not correlate with TTP. CONCLUSIONS Maintenance sunitinib was feasible but did not improve 6-ms progression rate; open label sunitinib had limited activity. sVEGFR-2 decreased on sunitinib.

Raman spectroscopy of bone metastasis

Raman spectroscopy of bone has been used to characterize chemical changes occurring in diseases such as osteoporosis, osteoarthritis and osteomyelitis. Metastasis of cancer into bone causes changes to bone quality that are similar to those observed in osteoporosis, such as decreased bone strength, but with an accelerated timeframe. In particular, osteolytic (bone degrading) lesions in bone metastasis have a marked effect on patient quality of life because of increased risk of fractures, pain, and hypercalcemia. We use Raman spectroscopy to examine bone from two different mouse models of osteolytic bone metastasis. Raman spectroscopy measures physicochemical information which cannot be obtained through standard biochemical and histological measurements. This study was reviewed and approved by the University of Michigan University Committee on the Care and Use of Animals. Two mouse models of prostate cancer bone metastasis, RM1 (n=3) and PC3-luc (n=4) were examined. Tibiae were injected with RM1 or PC3-luc cancer cells, while the contralateral tibiae received a placebo injection for use as controls. After 2 weeks of incubation, the mice were sacrificed and the tibiae were examined by Raman microspectroscopy (λ=785 nm). Spectroscopic markers corresponding to mineral stoichiometry, bone mineralization, and mineral crystallinity were compared in spectra from the cancerous and control tibiae. X-ray imaging of the tibia confirmed extensive osteolysis in the RM1 mice, with tumor invasion into adjoining soft tissue and moderate osteolysis in the PC3-luc mice. Raman spectroscopic markers indicate that osteolytic lesions are less mineralized than normal bone tissue, with an altered mineral stoichiometry and crystallinity.