Divya Sahu

A fragment of secreted Hsp90α carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice

Wounds that fail to heal in a timely manner, for example, diabetic foot ulcers, pose a health, economic, and social problem worldwide. For decades, conventional wisdom has pointed to growth factors as the main driving force of wound healing; thus, growth factors have become the center of therapeutic developments. To date, becaplermin (recombinant human PDGF-BB) is the only US FDA-approved growth factor therapy, and it shows modest efficacy, is costly, and has the potential to cause cancer in patients. Other molecules that drive wound healing have therefore been sought. In this context, it has been noticed that wounds do not heal without the participation of secreted Hsp90α. Here, we report that a 115-aa fragment of secreted Hsp90α (F-5) acts as an unconventional wound healing agent in mice. Topical application of F-5 peptide promoted acute and diabetic wound closure in mice far more effectively than did PDGF-BB. The stronger effect of F-5 was due to 3 properties not held by conventional growth factors: its ability to recruit both epidermal and dermal cells; the fact that its ability to promote dermal cell migration was not inhibited by TGF-β; and its ability to override the inhibitory effects of hyperglycemia on cell migration in diabetes. The discovery of F-5 challenges the long-standing paradigm of wound healing factors and reveals a potentially more effective and safer agent for healing acute and diabetic wounds.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates mitochondrial biogenesis and respiration via estrogen-related receptor α (ERRα).

Background: Aberrant PTEN/PI3K signaling and mitochondrial abnormalities are commonly associated with cancer. Results: PTEN loss and activation of PI3K/protein kinase B up-regulate ERRα, increase mitochondrial mass, and induce a metabolic pattern similar to the “Warburg effect.” Conclusion: PTEN/PI3K signaling controls mitochondrial mass and function by regulating ERRα through the AKT/CREB axis. Significance: This study establishes a novel link between oncogenic signaling and dysregulated mitochondrial metabolism. Mitochondrial abnormalities are associated with cancer development, yet how oncogenic signals affect mitochondrial functions has not been fully understood. In this study, we investigate the relationship between mitochondrial alterations and PI3K/protein kinase B (AKT) signaling activation using hepatocytes and liver tissues as our experimental models. We show here that liver-specific deletion of Pten, which leads to activation of PI3K/AKT, is associated with elevated oxidative stress, increased mitochondrial mass, and augmented respiration accompanied by enhanced glycolysis. Consistent with these observations, estrogen-related receptor α (ERRα), an orphan nuclear receptor known for its role in mitochondrial biogenesis, is up-regulated in the absence of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Our pharmacological and genetic studies show that PI3K/AKT activity regulates the expression of ERRα and mitochondrial biogenesis/respiration. Furthermore, cAMP-response element-binding protein, as a downstream target of AKT, plays a role in the regulation of ERRα, independent of PKA signaling. ERRα regulates reactive oxygen species production, and ERRα knockdown attenuates proliferation and colony-forming potential in Pten-null hepatocytes. Finally, analysis of clinical datasets from liver tissues showed a negative correlation between expressions of ERRα and PTEN in patients with liver cancer. Therefore, this study has established a previously unrecognized link between a growth signal and mitochondrial metabolism.

A potentially common peptide target in secreted heat shock protein-90α for hypoxia-inducible factor-1α-positive tumors.

ETOC: Deregulated/overexpressed HIF-1α is found in many solid tumors, and directly sabotaging it is challenging therapeutically. HIF-1α uses secreted Hsp90α, which uses a key epitope, F-5, for invasion and tumor formation. Drugs that target F-5 may be more effective and less toxic for treatment of HIF-1α–positive tumors.

Evolutionarily conserved dual lysine motif determines the non-chaperone function of secreted Hsp90alpha in tumour progression

Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (α and β) have been shown to support tumour progression. The tumour-supporting activity of the intracellular Hsp90 is attributed to their N-terminal ATPase-driven chaperone function. What molecular entity determines the extracellular function of secreted Hsp90 and the distinction between Hsp90α and Hsp90β was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90α nullifies tumour cells’ ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90β leads to tumour cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90β, protein recovers tumourigenicity of the Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90β subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90β with lysines convert Hsp90β to a Hsp90α-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α inhibits both de novo tumour formation and expansion of already formed tumours in mice. This study suggests an alternative therapeutic approach to target Hsp90 in cancer, that is, the tumour-secreted Hsp90α, instead of the intracellular Hsp90α and Hsp90β.

The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

Summary When skin is wounded, migration of epidermal keratinocytes at the wound edge initiates within hours, whereas migration of dermal fibroblasts toward the wounded area remains undetectable until several days later. This “cell type traffic” regulation ensures proper healing of the wound, as disruptions of the regulation could either cause delay of wound healing or result in hypertrophic scars. TGF&bgr;3 is the critical traffic controller that selectively halts migration of the dermal, but not epidermal, cells to ensure completion of wound re-epithelialization prior to wound remodeling. However, the mechanism of TGF&bgr;3s anti-motility signaling has never been investigated. We report here that activated T&bgr;RII transmits the anti-motility signal of TGF&bgr;3 in full to T&bgr;RI, since expression of the constitutively activated T&bgr;RI-TD mutant was sufficient to replace TGF&bgr;3 to block PDGF-bb-induced dermal fibroblast migration. Second, the three components of R-Smad complex are all required. Individual downregulation of Smad2, Smad3 or Smad4 prevented TGF&bgr;3 from inhibiting dermal fibroblast migration. Third, Protein Kinase Array allowed us to identify the protein kinase A (PKA) as a specific downstream effector of R-Smads in dermal fibroblasts. Activation of PKA alone blocked PDGF-bb-induced dermal fibroblast migration, just like TGF&bgr;3. Downregulation of PKAs catalytic subunit nullified the anti-motility signaling of TGF&bgr;3. This is the first report on anti-motility signaling mechanism by TGF&bgr; family cytokines. Significance of this finding is not only limited to wound healing but also to other human disorders, such as heart attack and cancer, where the diseased cells have often managed to avoid the anti-motility effect of TGF&bgr;.

Abstract 3913: Hypoxia-mediated cancer imaging by a novel class of near-infrared (NIR) heptamethine cyanine dyes.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Near-infrared (NIR) fluorescence imaging has great potential for noninvasive tumor detection. In this study, we describe a novel hypoxia-mediated mechanism underlying a group of heptamethine cyanine dyes, MHI-148 and IR-783, which we developed previously that show tumor-specific targeting properties in cancer cells and living animals (Yang et al., Clin Cancer Res 2010, 16:2833-44 and J Urol 2013, in press). Hypoxia is a common condition found in a wide range of tumors, and mediates many aspects of cancer progression at both tissue and single-cell levels. Using a hypoxia-sensitive luciferase reporter construct (5HREp-ODD-luc) to assess dynamic and real-time tumor hypoxia, in vivo and ex vivo dual-modality imaging studies of human prostate cancer (PCa) growth in nude mice indicate superimposed distribution of NIR signals with hypoxia in tumors. Hypoxia and hypoxic mimickers augment NIR dye uptake in multiple human and murine cell lines representing different types of cancer. We determined that the increased dye uptake under hypoxic conditions was mediated by hypoxia-inducible factor 1α (HIF1α) both in vitro and in vivo. Using cDNA microarray analysis, we identified a group of organic anion-transporting polypeptide (OATP) genes (e.g. OATP1B3, OATP2B1 and OATP5A1), which were highly inducible by hypoxia/HIF1α in PCa cells. We further showed increased co-expression of HIF1α and a representative OATP1B3 gene upon PCa progression in clinical specimens. Pharmacological inhibition or genetic silencing of the OATP1B3 gene reduced the uptake of NIR dyes in tumor cells and tissues. Moreover, hypoxia/HIF1α up-regulates the transcriptional and translational expression of OATP1B3 in PCa, and HIF1α directly binds to a hypoxia response element in the OATP1B3 promoter determined by chromatin immunoprecipitation assays. In addition, we applied NIR dyes for clinical detection of renal cell carcinomas, which shows consistent results with clinical diagnostic tests, and importantly, the HIF1α/OATP1B3 signaling was manifested in these tumors. Together, we provide for the first time the molecular mechanisms of specific NIR dye uptake in cancer via tumor hypoxia and HIF1α/OATPs signals, and present clinical evidence for its potential future applications in cancer detection, prognosis, and therapy. Citation Format: Jason Boyang Wu, Chen Shao, Xiangyan Li, Peizhen Hu, Yi-Ting Chen, Xiaoliang Dou, Divya Sahu, Wei Li, Hiroshi Harada, Ruoxiang Wang, Haiyen E. Zhau, Leland W.K. Chung. Hypoxia-mediated cancer imaging by a novel class of near-infrared (NIR) heptamethine cyanine dyes. [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 3913. doi:10.1158/1538-7445.AM2013-3913