Hidemi Saito

Increased Renal Calcium Reabsorption by Parathyroid Hormone–Related Protein Is a Causative Factor in the Development of Humoral Hypercalcemia of Malignancy Refractory to Osteoclastic Bone Resorption Inhibitors

Purpose: Bisphosphonate and calcitonin lower blood calcium in humoral hypercalcemia of malignancy (HHM) by suppressing osteoclastic bone resorption, but repeated administration of these drugs often leads to relapse. In this study, we examined the roles of parathyroid hormone–related protein (PTHrP) in the development of bisphosphonate- and calcitonin-refractory HHM. Experimental Design: Nude rats bearing the LC-6 JCK tumor xenograft (LC-6 rats) exhibited high bone turnover and HHM. Repeated administration of alendronate induced a sustained suppression of the bone resorption, but it caused only early and transient reduction of the blood calcium levels, leading to unresponsiveness to the drug. Because high blood levels of PTHrP were detected in the LC-6 rats, those that developed alendronate-refractory HHM were treated with an anti-PTHrP antibody. Results: Administration of anti-PTHrP antibody to animals that received repeated administration of alendronate, thereby developing alendronate-refractory HHM, resulted in an increase in fractional excretion of calcium and a marked decrease of blood calcium level. Drug-refractory HHM was also observed in animals that received another osteoclast inhibitor, an eel calcitonin analogue elcatonin. The blood calcium level decreased after the initial administration of elcatonin, but it eventually became elevated during repeated administration. Administration of the anti-PTHrP antibody, but not of alendronate, effectively reduced the blood calcium of the animals that developed elcatonin-refractory HHM. Conclusion: High levels of circulating PTHrP and the resulting augmentation of renal calcium reabsorption is one of the major causes of the emergence of osteoclast inhibitor-refractory HHM. Thus, blockage of PTHrP functions by a neutralizing antibody against PTHrP would benefit patients who develop bisphosphonate- or calcitonin-refractory HHM.

Parathyroid hormone–related protein (PTHrP) as a causative factor of cancer-associated wasting: Possible involvement of PTHrP in the repression of locomotor activity in rats bearing human tumor xenografts

Nude rats bearing the LC‐6‐JCK human lung cancer xenograft displayed cancer‐associated wasting syndrome in addition to humoral hypercalcemia of malignancy. In these rats, not only PTHrP but also several other human proinflammatory cytokines, such as IL‐6, leukemia‐inducing factor, IL‐8, IL‐5 and IL‐11, were secreted to the bloodstream. Proinflammatory cytokines induce acute‐phase reactions, as evidenced by a decrease of serum albumin and an increase in α1‐acid glycoprotein. Tumor resection abolished the production of proinflammatory cytokines and improved acute‐phase reactions, whereas anti‐PTHrP antibody affected neither proinflammatory cytokine production nor acute‐phase reactions. Nevertheless, tumor resection and administration of anti‐PTHrP antibody similarly and markedly attenuated not only hypercalcemia but also loss of fat, muscle and body weight. Body weight gain by anti‐PTHrP antibody was associated with increased food consumption; increased body weight from anti‐PTHrP antibody was observed when animals were freely fed but not when they were given the same feeding as those that received only vehicle. Furthermore, nude rats bearing LC‐6‐JCK showed reduced locomotor activity, less eating and drinking and low blood phosphorus; and anti‐PTHrP antibody restored them. Although alendronate, a bisphosphonate drug, decreased blood calcium, it affected neither locomotor activity nor serum phosphorus level. These results indicate that PTHrP represses physical activity and energy metabolism independently of hypercalcemia and proinflammatory cytokine actions and that deregulation of such physiologic activities and functions by PTHrP is at least in part involved in PTHrP‐induced wasting syndrome.

Involvement of cyclooxygenase-2 in the tumor site-dependent production of parathyroid hormone-related protein in colon 26 carcinoma

It has been shown that in the mouse colon 26 tumor model, tumors grown in the subcutis (subcutis colon 26) caused early onset of cachectic syndromes, whereas those in the liver (liver colon 26) did not. Both interleukin (IL)‐6 and parathyroid hormone‐related protein (PTHrP) were involved in the development of cachectic syndromes in this tumor model. However, whether expression of PTHrP and IL‐6 is differently regulated in the tumor microenvironment is unclear. In the present study, culturing the colon 26 cells under different conditions in vitro revealed that IL‐6 production was increased by monolayer culture under a low‐glucose condition but not by spheroid culture. In contrast, PTHrP production was increased by spheroid culture but not by monolayer culture, even under a low‐glucose condition. Gene expression profiling revealed that the expression of cyclooxygenase (COX)‐2 was up‐regulated in both subcutis colon 26 and spheroid cultures, and that COX‐2 inhibitor NS‐398 suppressed PTHrP production in spheroid cultures. Furthermore, administration of NS‐398 decreased the PTHrP level without affecting the tumor growth in mice bearing subcutis colon 26. These results demonstrate that production of PTHrP and IL‐6 largely depends on the microenvironments in which tumors are developed or metastasized and that up‐regulation of COX‐2 in a necrobiotic environment leads to PTHrP production, thereby causing cachectic syndromes. (Cancer Sci 2007; 98: 1563–1569)

Abstract 2524: Targeting microtubule-associated protein 4 with novel small molecule CH4938056 confers antitumor activity under quasi in vivo conditions and in xenograft models

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tumor microenvironment is a major factor influencing treatment resistance to conventional anticancer therapies. Indeed, under “quasi in vivo” conditions, with low oxygen (1%), low pH (6.5) and low glucose concentration (0.01%) to mimic the environments in grafted tumors in mice or in clinical tumors, anti-proliferative activities of some conventional anticancer agents were diminished. Here, we describe the small molecule CH4938056, which has a novel chemical structure and was identified through cell-based screening under the “quasi in vivo” conditions followed by chemical modification. Our initial phenotypic profiling revealed that CH4938056 specifically arrests cells at the M phase and that it overcomes multiple resistance mechanisms to conventional anticancer agents including over-expression of MDR1 and BCRP. Then, after designing and identifying a water-soluble phosphate prodrug which successfully converts to CH4938056 after injection, we demonstrated antitumor efficacy of CH4938056 in a HCT116 xenograft model and a MDR1-overexpressing cancer model. Chemo-proteomic studies and consequent biochemical analysis revealed that CH4938056 binds to microtubule-associated protein 4 (MAP4). Moreover, CH4938056 inhibited MAP4-dependent microtubule assembly in a cell free system. siRNA-mediated knockdown of MAP4 induced chromosomal misalignment in metaphase cells, which closely resembles the primary phenotype of the CH4938056-treated cells. Under the “quasi in vivo” conditions, MAP4 expression turned out to be down-regulated (since MAP4 transcription is known to be negatively regulated by p53 which is up-regulated under these conditions) and when we knocked down MAP4 with siRNA, cancer cells became sensitive to CH4938056, which altogether is consistent with the fact that CH4935056 has antitumor activity even under the “quasi in vivo” conditions. From these observations, we conclude that CH4938056 inhibits proliferation of cancer cells by targeting MAP4. Targeting MAP4 with CH4938056 offers a novel approach for the treatment of cancer, especially for patients resistant to conventional anticancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2011-2524