IntroductionThe human epidermal growth factor receptor 2 (HER2)-targeted therapies trastuzumab (T) and lapatinib (L) show high efficacy in patients with HER2-positive breast cancer, but resistance is prevalent. Here we investigate resistance mechanisms to each drug alone, or to their combination using a large panel of HER2-positive cell lines made resistant to these drugs.MethodsResponse to L + T treatment was characterized in a panel of 13 HER2-positive cell lines to identify lines that were de novo resistant. Acquired resistant lines were then established by long-term exposure to increasing drug concentrations. Levels and activity of HER2 and estrogen receptor (ER) pathways were determined by qRT-PCR, immunohistochemistry, and immunoblotting assays. Cell growth, proliferation, and apoptosis in parental cells and resistant derivatives were assessed in response to inhibition of HER or ER pathways, either pharmacologically (L, T, L + T, or fulvestrant) or by using siRNAs. Efficacy of combined endocrine and anti-HER2 therapies was studied in vivo using UACC-812 xenografts.ResultsER or its downstream products increased in four out of the five ER+/HER2+ lines, and was evident in one of the two intrinsically resistant lines. In UACC-812 and BT474 parental and resistant derivatives, HER2 inhibition by T reactivated HER network activity to promote resistance. T-resistant lines remained sensitive to HER2 inhibition by either L or HER2 siRNA. With more complete HER2 blockade, resistance to L-containing regimens required the activation of a redundant survival pathway, ER, which was up-regulated and promoted survival via various Bcl2 family members. These L- and L + T-resistant lines were responsive to fulvestrant and to ER siRNA. However, after prolonged treatment with L, but not L + T, BT474 cells switched from depending on ER as a survival pathway, to relying again on the HER network (increased HER2, HER3, and receptor ligands) to overcome L's effects. The combination of endocrine and L + T HER2-targeted therapies achieved complete tumor regression and prevented development of resistance in UACC-812 xenografts.ConclusionsCombined L + T treatment provides a more complete and stable inhibition of the HER network. With sustained HER2 inhibition, ER functions as a key escape/survival pathway in ER-positive/HER2-positive cells. Complete blockade of the HER network, together with ER inhibition, may provide optimal therapy in selected patients.
Fibroblast growth factor 23 (FGF23) is a phosphaturic and vitamin D-regulatory hormone of putative bone origin that is elevated in patients with chronic kidney disease (CKD). The mechanisms responsible for elevations of FGF23 and its role in the pathogenesis of chronic kidney disease-mineral bone disorder (CKD-MBD) remain uncertain. We investigated the association between FGF23 serum levels and kidney disease progression, as well as the phenotypic features of CKD-MBD in a Col4a3 null mouse model of human autosomal-recessive Alport syndrome. These mice exhibited progressive renal failure, declining 1,25(OH)2D levels, increments in PTH and FGF23, late onset hypocalcemia and hyperphosphatemia, high-turnover bone disease, and increased mortality. Serum levels of FGF23 increased in the earliest stages of renal damage, prior to elevations in BUN and creatinine. FGF23 gene transcription in bone, however, did not increase until late-stage kidney disease, when serum FGF23 levels were exponentially elevated. Further evaluation of bone revealed trabecular osteocytes to be the primary cell source for FGF23 production in late-stage disease. Changes in FGF23 mirrored the rise in serum PTH and the decline in circulating 1,25(OH) 2D. The rise in PTH and FGF23 in Col4a3 null mice coincided with an increase in the urinary fractional excretion of phosphorus and a progressive decline in sodium-phosphate co-transporter gene expression in the kidney. Our findings suggest elevations of FGF23 in CKD to be an early marker of renal injury that increases prior to BUN and serum creatinine. An increased production of FGF23 by bone may not be responsible for early increments in FGF23 in CKD, but does appear to contribute to FGF23 levels in late-stage disease. Elevations in FGF23 and PTH coincide with an increase in urinary phosphate excretion that likely prevents the early onset of hyperphosphatemia in the face of increased bone turnover and a progressive decline in functional renal mass.
Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone that in end-stage renal disease is markedly increased in serum; however, the mechanisms responsible for this increase are unclear. Here, we tested whether phosphate retention in chronic kidney disease (CKD) is responsible for the elevation of FGF23 in serum using Col4α3 knockout mice, a murine model of Alport disease exhibiting CKD. We found a significant elevation in serum FGF23 in progressively azotemic 8 and 12 week-old CKD mice along with an increased fractional excretion of phosphorus. Both moderate and severe phosphate restriction reduced fractional excretion of phosphorus by 8 weeks, yet serum FGF23 levels remained strikingly elevated. By 12 weeks, FGF23 levels were further increased with moderate phosphate restriction, while severe phosphate restriction led to severe bone mineralization defects and decreased FGF23 production in bone. CKD mice on a control diet had low serum 1,25(OH)2D levels and 3-fold higher renal Cyp24α1 gene expression compared to wild-type mice. Severe phosphate restriction increased 1,25(OH)2D levels in CKD mice by 8 weeks and lowered renal Cyp24α1 gene expression despite persistently elevated serum FGF23. Renal klotho gene expression declined in CKD mice on a control diet, but improved with severe phosphate restriction. Thus, dietary phosphate restriction reduces the fractional excretion of phosphorus independent of serum FGF23 levels in mice with CKD.
The coregulator steroid receptor coactivator (SRC)-1 increases transcriptional activity of the estrogen receptor (ER) in a number of tissues including bone. Mice deficient in SRC-1 are osteopenic and display skeletal resistance to estrogen treatment. SRC-1 is also known to modulate effects of selective ER modulators like tamoxifen. We hypothesized that single nucleotide polymorphisms (SNP) in SRC-1 may impact estrogen and/or tamoxifen action. Because the only nonsynonymous SNP in SRC-1 (rs1804645; P1272S) is located in an activation domain, it was examined for effects on estrogen and tamoxifen action. SRC-1 P1272S showed a decreased ability to coactivate ER compared with wild-type SRC-1 in multiple cell lines. Paradoxically, SRC-1 P1272S had an increased protein half-life. The Pro to Ser change disrupts a putative glycogen synthase 3 (GSK3)β phosphorylation site that was confirmed by in vitro kinase assays. Finally, knockdown of GSK3β increased SRC-1 protein levels, mimicking the loss of phosphorylation at P1272S. These findings are similar to the GSK3β-mediated phospho-ubiquitin clock previously described for the related coregulator SRC-3. To assess the potential clinical significance of this SNP, we examined whether there was an association between SRC-1 P1272S and selective ER modulators response in bone. SRC-1 P1272S was associated with a decrease in hip and lumbar bone mineral density in women receiving tamoxifen treatment, supporting our in vitro findings for decreased ER coactivation. In summary, we have identified a functional genetic variant of SRC-1 with decreased activity, resulting, at least in part, from the loss of a GSK3β phosphorylation site, which was also associated with decreased bone mineral density in tamoxifen-treated women.
Solid organ and stem cell transplant recipients have an increased risk of developing cutaneous infections, which often are refractory to conventional treatment (Euvrard et al., Journal of the American Academy of Dermatology, 2001, 44, 932–939). Molluscum contagiosum, a common self‐limited disease primarily affecting children, can be more severe and unresponsive to therapy in transplant patients (Gardner & Ormond, Clinical and Experimental Dermatology, 2006, 31, 452–453). Candida immunotherapy has been widely used for the treatment of warts, and recently its application has been expanded to include treatment of symptomatic molluscum in pediatric patients (Enns & Evans, Pediatric Dermatology, 2011, 28, 254–258; Maronn et al., Pediatric Dermatology, 2008, 25, 189–192). However, to our knowledge there have been no reports in the literature of its utility in the setting of adult transplant or immunocompromised patients. Herein, we report a case of successful treatment of refractory molluscum contagiosum in a stem cell transplant patient with Candida immunotherapy.
We describe a case of compensatory vasodilation in the vasculature surrounding the site of long-term brimonidine use for the treatment of rosacea.Report of a Case | A 57-year-old woman with a medical history of rosacea, anxiety, hyperlipidemia, and hypertension presented with persistent, patchy erythema on the lateral face and neck of several weeks' duration. Seven months before presentation, she had been prescribed brimonidine, 0.33%, topical gel for persistent facial erythema secondary to rosacea. She reported appropriate use of the medication, applying the prescribed amount only to affected areas on her central face. Physical examination revealed marked bright erythema diffusely covering areas of the lateral cheeks, neck, and upper chest (Figure, A). Interestingly, there was clear sparing of the sites of brimonidine application on the central face.Review of systems revealed only that the eruption was associated with mild burning. Prior to presenting to our clinic, she saw several other dermatologists. Over the course of these visits, several laboratory studies were ordered, including a complete blood cell count with differential, a complete metabolic panel, a lipid panel, and measures of C1, total complement, antinuclear antibody (ANA), and tryptase, all of which yielded normal results. A cutaneous punch biopsy of the left neck was performed and revealed nonspecific findings of photodamaged skin with telangiectasias and scant perivascular lymphocytic inflammation with no specific evidence of an autoimmune etiology. The patient was advised to discontinue using the brimonidine gel and to follow up in clinic in 1 week. At 1-week follow-up, her symptoms improved dramatically (Figure, B), leading us to believe that her initial reaction was secondary to long-term brimonidine use.Discussion | Since its approval by the US Food and Drug Administration in 2013, topical brimonidine gel has offered effective therapy for certain patients with persistent facial erythema secondary to rosacea.1 Previous studies reveal that most patients tolerate this therapy quite well without any adverse reactions. 2 However, there have been reports of cutaneous adverse reactions at the site of brimonidine application. These include flushing, worsening erythema, burning sensation, and contact dermatitis, most of which present immediately or early in the course of therapy. 3 Herein, we describe a patient who had been using brimonidine for nearly 7 months before developing what we believe to be a novel adverse drug reaction. Interestingly, our patient's symptoms were in areas surrounding the site of brimonidine use, sparing the tissue in direct contact with the gel. Brimonidine is a relatively selective α2 adrenergic agonist. Topical application causes vasoconstriction of superficial vessels at the site of application, allowing for the reduction of erythema. We hypothesize that the reaction seen in our patient represents a compensatory vasodilation of vessels in the surrounding skin due to chronic vasoconstriction at the site of long-term bri...
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