Deregulated accumulation of hypoxia-inducible factor-1α (HIF-1α) is a hallmark of many solid tumors. Directly targeting HIF-1α for therapeutics is challenging. Our finding that HIF-1α regulates secretion of heat shock protein-90α (Hsp90α) for cell migration raises the exciting possibility that targeting the secreted Hsp90α from HIF-1α-positive tumors has a better clinical outlook. Using the HIF-1α-positive and metastatic breast cancer cells MDA-MB-231, we show that down-regulation of the deregulated HIF-1α blocks Hsp90α secretion and invasion of the cells. Reintroducing an active, but not an inactive, HIF-1α into endogenous HIF-1α-depleted cells rescues both Hsp90α secretion and invasion. Inhibition of Hsp90α secretion, neutralization of secreted Hsp90α action, or removal of the cell surface LRP-1 receptor for secreted Hsp90α reduces the tumor cell invasion in vitro and lung colonization and tumor formation in nude mice. Furthermore, we localized the tumor-promoting effect to a 115-amino acid region in secreted Hsp90α called F-5. Supplementation with F-5 is sufficient to bypass the blockade of HIF-1α depletion and resumes invasion by the tumor cells under serum-free conditions. Because normal cells do not secrete Hsp90α in the absence of stress, drugs that target F-5 should be more effective and less toxic in treatment of HIF-1α-positive tumors in humans.

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. TGFβ3 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 TGFβ3's anti-motility signaling has never been investigated. We report here that activated TβRII transmits the anti-motility signal of TGFβ3 in full to TβRI, since expression of the constitutively activated TβRI-TD mutant was sufficient to replace TGFβ3 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 TGFβ3 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 TGFβ3. Downregulation of PKA's catalytic subunit nullified the anti-motility signaling of TGFβ3. This is the first report on anti-motility signaling mechanism by TGFβ 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 TGFβ.

Tumor cells face constant stress of ischemic (nutrient paucity and hypoxia) environment when they migrate and invade too fast to outgrow the nearest blood vessels. During the temporary loss of support from circulation, the tumor cells must act self-sufficient to survive and then to migrate to re-connect with the nearest blood supply or die. We have previously reported that ablation of the low-density lipoprotein receptor-related protein 1 (LRP-1) completely nullified the ability of tumour cells to migrate and invade under serum-free conditions in vitro and to form tumours in vivo. The mechanism behind the important function by cell surface LRP-1 was not fully understood. Herein we show that LRP-1 orchestrates two parallel cell surface signalling pathways to support the full constitutive tumour cell migration. First, LRP-1 stabilizes activated epidermal growth factor receptor (EGFR) to contribute half of the pro-motility signalling. Second, LRP-1 mediates secreted Hsp90α autocrine signalling to bring the other half of pro-motility signalling. Only combined inhibitions of the EGFR signalling and the eHsp90α autocrine signalling led to the full blockade of the tumour cell migration as the LRP-1 depletion did. This finding uncovers a novel mechanism by which certain breast cancer cells use LRP-1 to engage parallel signalling pathways to move when they lose contact with blood support.

Extracellular heat shock protein-90alpha (eHsp90α) plays an essential role in tumour invasion and metastasis. The plasma eHsp90α levels in patients with various cancers correlate with the stages of the diseases. Nonetheless, the mechanism of action by tumour-secreted eHsp90α remained unclear. Here we show that eHsp90α accounts for approximately 1% of the total cellular Hsp90α and is associated with tumour-secreted exosomes. CRISPR-cas9 knockout of Hsp90α did not affect the overall distribution and quantity of secreted exosomes, but it caused increased exosome-associated CD9 and decreased exosome-associated TSG101, Alix, and CD63. However, Hsp90α-knockout tumour cells have not only lost their own constitutive motility, but also the ability to recruit stromal cells via secreted exosomes. These defects are specifically due to the lack of eHsp90α on tumour cell-secreted exosomes. Anti-Hsp90α antibody nullified the pro-motility activity of tumour-secreted exosomes and human recombinant Hsp90α protein fully rescued the functional defects of eHsp90α-free exosomes. Finally, while current exosome biogenesis models exclusively implicate the luminal location of host cytosolic proteins inside secreted exosomes, we provide evidence for eHsp90α location on the external surface of tumour-secreted exosomes. Taken together, this study elucidates a new mechanism of action by exosome-associated eHsp90α.

Burn injuries are a leading cause of morbidity including prolonged hospitalization, disfigurement, and disability. Currently there is no Food and Drug Administration-approved burn therapeutics. A clinical distinction of burn injuries from other acute wounds is the event of the so-called secondary burn wound progression within the first week of the injury, in which a burn expands horizontally and vertically from its initial boundary to a larger area. Therefore, an effective therapeutics for burns should show dual abilities to prevent the burn wound progression and thereafter promote burn wound healing. Herein we report that topically applied F-5 fragment of heat shock protein-90α is a dual functional agent to promote burn wound healing in pigs. First, F-5 prevents burn wound progression by protecting the surrounding cells from undergoing heat-induced caspase 3 activation and apoptosis with increased Akt activation. Accordingly, F-5-treated burn and excision wounds show a marked decline in inflammation. Thereafter, F-5 accelerates burn wound healing by stimulating the keratinocyte migration-led reepithelialization, leading to wound closure. This study addresses a topical agent that is capable of preventing burn wound progression and accelerating burn wound healing.

Rapidly growing tumours in vivo often outgrow their surrounding available blood supply, subjecting themselves to a severely hypoxic microenvironment. Understanding how tumour cells adapt themselves to survive hypoxia may help to develop new treatments of the tumours. Given the limited blood perfusion to the enlarging tumour, whatever factor(s) that allows the tumour cells to survive likely comes from the tumour cells themselves or its associated stromal cells. In this report, we show that HIF-1α-overexpressing breast cancer cells, MDA-MB-231, secrete heat shock protein-90alpha (Hsp90α) and use it to survive under hypoxia. Depletion of Hsp90α secretion from the tumour cells was permissive to cytotoxicity by hypoxia, whereas supplementation of Hsp90α-knockout tumour cells with recombinant Hsp90α, but not Hsp90β, protein prevented hypoxia-induced cell death via an autocrine mechanism through the LDL receptor-related protein-1 (LRP1) receptor. Finally, direct inhibition of the secreted Hsp90α with monoclonal antibody, 1G6-D7, enhanced tumour cell death under hypoxia. Therefore, secreted Hsp90α is a novel survival factor for certain tumours under hypoxia.

Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) beta3 levels, which are undetectable in plasma and high in serum, and by TGFbeta receptor (TbetaR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFbeta3 from serum converts serum to a plasmalike reagent. The addition of TGFbeta3 to plasma converts it to a serumlike reagent. Down-regulation of TbetaRII in dermal cells or up-regulation of TbetaRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma-->serum-->plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have severe, incurable skin fragility, blistering, and multiple skin wounds due to mutations in the gene encoding type VII collagen (C7), the major component of anchoring fibrils mediating epidermal-dermal adherence. Nearly 10-25% of RDEB patients carry nonsense mutations leading to premature stop codons (PTCs) that result in truncated C7. In this study, we evaluated the feasibility of using aminoglycosides to suppress PTCs and induce C7 expression in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906) and two primary RDEB fibroblasts (R578X/R578X and R163X/R1683X). Incubation of these cells with aminoglycosides (geneticin, gentamicin, and paromomycin) resulted in the synthesis and secretion of a full-length C7 in a dose-dependent and sustained manner. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction of skin equivalents. We further demonstrated the general utility of aminoglycoside-mediated readthrough in 293 cells transiently transfected with expression vectors encoding 22 different RDEB nonsense mutations. This is the first study demonstrating that aminoglycosides can induce PTC readthrough and restore functional C7 in RDEB caused by nonsense mutations. Therefore, aminoglycosides may have therapeutic potential for RDEB patients and other inherited skin diseases caused by nonsense mutations.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.